title: FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

city: Solna

first: Andrä

last: Brunner

address: Solnavägen 9

department: Department of Cell and Molecular Biology

country: Sweden

institute: Karolinska Institutet

summary: Oncogene-induced replication stress constitutes an early obstacle for pre-cancerous cells to overcome to progress towards malignancy. Fanconi anaemia signalling represents a major genomic maintenance pathway that is activated in response to replication stress, impinging on stalled replication fork stability and recovery. Here, we report that upon replication stress, phosphorylation of the FANCD2 N-terminus by CHK1 triggers FBXL12-dependent proteasomal degradation of FANCD2, facilitating clearance of FANCD2 at stalled replication forks. This mechanism is required to promote efficient and faithful DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Notably, reconstitution of FANCD2 with mutations in the N-terminal phosphodegron fail to re-establish fork progression in FANCD2-deficient human fibroblasts in response to replication stress. In the absence of FBXL12, FANCD2 becomes trapped on chromatin leading to replication stress, excessive DNA damage, and cell death. In human cancers, FBXL12, CYCLIN E, and Fanconi anaemia signalling are positively correlated and upregulation or amplification of FBXL12 is linked to reduced survival in patients with high CYCLIN E expressing breast tumours. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitised breast cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability of CYCLIN E-overexpressing cancer cells and may represent a novel target for cancer therapy.

overall_design: Comparative gene expression profiling analysis of RNA-seq data obtained using MDA-MB-231 basal-like breast cancer cells. Triplicate samples from FBXL12-WT (transduced with scrambled control gRNA) reference cells and two FBXL12-KO clones were profiled.

first: Qiuzhen

last: Li

first: Samuele

last: Fisicaro

first: Alexandros

last: Kourtesakis

first: Johanna

last: Viiliäinen

first: Henrik

last: Johansson

first: Vijaya

last: Pandey

first: Asarsh

last: Mayank

first: Janne

last: Lehtiö

first: James

last: Wohlschlegel

first: Charles

last: Spruck

first: Juha

last: Rantala

first: Lukas

last: Orre

first: Olle

last: Sangfelt


title: Regulators of mitonuclear balance link mitochondrial metabolism to mtDNA expression

status: Public on Aug 17 2023

city: Boston

first: Nicholas

middle: J

last: Kramer

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

country: USA

institute: Harvard Medical School

summary: Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells requiring coordinated gene expression across organelles. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of Complex IV. We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically impacts Complex IV biogenesis by acting at the intersection of mitochondrial lipid metabolism and protein synthesis, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 forms a complex with RCC1L, which together perform NDPK activity to maintain local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, NME6 modulates the activity of mitoribosome regulatory complexes, altering mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression.

overall_design: sgRNA sequencing for CRISPR screens designed to detect mitonuclear balance regulators

first: Gyan

last: Prakash

first: Karine

last: Choquet

first: Iliana

last: Soto

first: Boryana

last: Petrova

first: Naama

last: Kanarek

first: L

middle: S

last: Churchman


title: RNA-seq analysis in RPMI-8402 cells after knockout of MYC and MYCN

status: Public on Aug 16 2023

city: Singapore

first: Tze King

last: Tan

address: 14 Medical Drive

department: Cancer Science Institute

country: Singapore

institute: National University of Singapore

type: Expression profiling by high throughput sequencing

summary: RNA-seq analysis was performed in a RPMI-8402 T-ALL cell stably expressing Cas9 aafter knockout of both MYC and MYCN

overall_design: RPMI-8402 cells stably expressing Cas9 were treated with either DMSO (Control) or Doxycline to induce expression of sgRNAs against MYC and MYCN for 72 hours.   Cells were then harvested and total RNA were extracted for library preparation and RNA-Seq analysis

first: Shi

middle: H

first: Tze

middle: K

first: Takaomi

last: Sanda


title: RNA-seq analysis in RPMI-8402 cells after knockout of MYCN

summary: RNA-seq analysis was performed in a RPMI-8402 T-ALL cell stably expressing Cas9 after knockout of MYCN

overall_design: RPMI-8402 cells stably expressing Cas9 were treated with either DMSO (Control) or Doxycline to induce expression of sgRNAs against MYCN for 72 hours.   Cells were then harvested and total RNA were extracted for library preparation and RNA-Seq analysis


title: E. coli Nissle 1917 transcriptional response after treatment with Calprotectin

city: Chicago

first: Nathan

last: Waldeck

state: IL

address: 303 E Superior St, Lurie 7-220

institute: Northwestern University

summary: To determine whether calprotectin can elicit any transcriptional response in the probiotic E. coli Nissle 1917(EcN), EcN was treated with 200 ug/g of calprotectin in log phase.

overall_design: We  performed gene expression profiling analysis using data obtained from RNA-seq of E. coli Nissle 1917 treated with 200 ug/g of calprotectin in log phase in both rich (LB) and minimal (M9) media conditions.

first: Jonathan

last: Xia

first: Arthur

last: Prindle


title: Cholesterol 24-hydroxylase at the choroid plexus restrains local inflammation and protects overall brain function

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Inflammation of the choroid plexus (CP), the interface between blood and cerebrospinal fluid, impacts the brain in aging and disease. Here, we report that the CP epithelium in mice and humans expresses CYP46A1 (cholesterol 24-hydroxylase), a brain-specific enzyme. CP CYP46A1 expression levels were found to be reduced in a mouse model of amyloidosis (5xFAD) and in aged mice, as well as in postmortem samples from COVID-19 victims. Bulk RNA-sequencing revealed that the CP epithelium responds in vitro to the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, by downregulating inflammatory transcriptomic signatures that we found in silico to be expressed in the CP across multiple neurological conditions. Moreover, the inflammatory cytokine TNF-α reduced CP Cyp46a1 expression, both in vitro and ex vivo, whereas Cyp46a1 overexpression attenuated the CP response to TNF-α. We propose that CYP46A1 plays a key regulatory role in CP homeostasis, with implications for various CNS pathologies, including sequelae of viral infections.

overall_design: Transcriptional profiling of choroid plexus epithelium cell cultures treated with DMSO/24-OH.

first: Afroditi

last: Tsitsou-Kampeli

first: Stefano

last: Suzzi

first: Mor

last: Kenigsbuch

first: Romano

last: Strobelt

first: Oded

last: Singer

first: Michal

last: Arad

first: Tamar

last: Ziv

first: Sarah

middle: P

last: Colaiuta

first: Eyal

last: David

first: Liora

last: Cahalon

first: Sedi

last: Medina

first: Denise

last: Kviatcovsky

first: Alexander

last: Kertser

first: Yosef

last: Shaul

last: Schwartz


title: TAL1 hijacks MYCN enhancer that induces MYCN expression and dependence on mevalonate pathway in T-cell acute lymphoblastic leukemia

first: Tze King

last: Tan

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: RNA-seq analysis in RPMI-8402 cells after knockout of MYC

summary: RNA-seq analysis was performed in a RPMI-8402 T-ALL cell stably expressing Cas9 after knockout of MYC

overall_design: RPMI-8402 cells stably expressing Cas9 were treated with either DMSO (Control) or Doxycline to induce expression of sgRNAs against MYC for 72 hours.   Cells were then harvested and total RNA were extracted for library preparation and RNA-Seq analysis

first: Shi

first: Tze

first: Takaomi

last: Sanda


title: Effect of acute pyrvinium pamoate treatment on gene expression of CD4+ T cells during early activation.

first: Kiran

last: Kurmi

address: Department of Cell Biology, Harvard Medical School, LHRRB, Room 314, 240 Longwood Avenue

department: Cell Biology

summary:
Treatment of T cells with pyrvinium pamoate (PP) early during their activation blocks mitochondrial biogenesis and results in reduced proliferation, skewed CD4+ T cell differentiation and reduced cytokine production.
Therefore, to examine the role of PP treatment on mitochondria health, we performed gene expression analysis to identify differentially expressed genes in naive CD4+ T cells cultured with anti-CD3/CD28 plus PP or DMSO. Naive CD4+ T cells were activated for six hours with 2 mg/mL anti-CD3/anti-CD28 mAbs and activated for an additional one hour in the presence or absence of PP.

overall_design: Three separate mice were used to isolate CD4+ T cells, and approx. 1x10^6 cells were used for each sample. DMSO (x3), 2 nM pyrvinium pamoate (x3), or 25 nM rotenone (x3) was used to examine their immediate effect on gene expression. Rotenone, which targets mitochondria, was used as an additional control.

first: Marcia

middle: C

last: Haigis


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [TT-seq]

city: London

first: Nick

last: Crump

address: Du Cane Road

department: Dept of Immunology and Inflammation

country: United Kingdom

institute: Imperial College London

summary: Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT and loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia, but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.

overall_design: TT-seq

first: Nicholas

middle: T

first: Alastair

middle: L

last: Smith

first: Thomas

middle: A

last: Milne


title: Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina II

status: Public on Aug 15 2023

city: San Francisco

first: Li Xuan

state: CA

address: 10 Koret Way

department: Ophthalmology

institute: UCSF

summary: Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet, how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) is essential for healthy vision. Disrupted autophagy due to mTOR overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting LC3, which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, TRPML1 (transient receptor potential-mucolipin 1)-dependent manner. RNAseq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment, and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these critical clearance mechanisms would help safeguard metabolic fitness of the RPE through the organismal lifespan.

overall_design: To investigate transcriptomic changes in WT and Abca4-/- mouse RPE, we collected total RNA and performed bulk RNA sequencing. Samples were collected at 5 h after light onset. 3 mice (replicates) were included for each genotype.


title: Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina


title: Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.

city: Mannheim

first: Francesca

last: Tuorto

address: Ludolf-Krehl-Str. 13-17

department: Division of Biochemistry  Mannheim Institute for Innate Immunoscience (MI3)

country: Germany

institute: Deutsches Krebsforschungszentrum

type: Non-coding RNA profiling by high throughput sequencing

summary:
Queuosine (Q) is a modified nucleoside at the wobble position of
specific tRNAs. In mammals, queuosinylation is facilitated by
queuine uptake from the gut microbiota and is introduced into
tRNA by the QTRT1-QTRT2 enzyme complex. By establishing a
Qtrt1 knockout mouse model, we discovered that the loss of QtRNA
leads to learning and memory deficits. Ribo-Seq analysis in
the hippocampus of Qtrt1-deficient mice revealed not only stalling
of ribosomes on Q-decoded codons but also a global imbalance in
translation elongation speed between codons that engage in weak
and strong interactions with their cognate anticodons. While Qdependent
molecular and behavioral phenotypes were identified in
both sexes, female mice were affected more severely than males.
Proteomics analysis confirmed deregulation of synaptogenesis and
neuronal morphology. Together, our findings provide a link
between tRNA modification and brain functions and reveal an
unexpected role of protein synthesis in sex-dependent cognitive
performance.

overall_design: Cytoplasmic RNA and Cytoplasmic RNA protected by ribosomes from RNase I (Ribonuclease I) cleavage, were sequenced. Small RNA libraries were performed according to the protocol using NEB NEXT Small RNA library Prep Set for Illumina (Multiplex Compatible) E7330, and size-selected (120-170 base pairs) before sequencing.

first: Cansu

last: Cirzi

first: Carine

last: Legrand

first: Johanna

last: Schott


title: A bacterial histone binds DNA in an unorthodox fashion

first: Tobias

last: Warnecke

institute: Imperial College

summary: Histones are the primary building blocks of chromatin in eukaryotes and many archaea. Bacteria are thought to rely on an orthogonal set of proteins to organize their chromosomes. Several bacterial genomes do, however, encode proteins with putative histone fold domains. Whether these proteins adopt a bona fide histone fold, assemble into higher order complexes that bind DNA, and play a central role in bacterial nucleoid physiology is not known. Here, we demonstrate that histones are major and essential building blocks of chromatin in the predatory bacterium Bdellovibrio bacteriovorus and the human pathogen Leptospira interrogans and likely important in several other bacterial clades. We determine the crystal structure of the B. bacteriovorus histone (Bd0055) dimer at 1.8Å resolution to reveal that histone fold topology, handshake dimer conformation, and the RD clamp motif are conserved between bacteria, archaea, and eukaryotes. However, ostensibly minor differences, including a shorter α2 helix, a less structured α3 helix, and a more acidic surface on one side of the dimer lead to a radically divergent DNA binding mode: instead of wrapping around the outer surface of a multi-subunit histone complex, DNA forms straight fibers, encased by a sheath of tightly packed Bd0055 dimers. Our results demonstrate that bacterial histones have evolved an atypical mode of DNA binding to become integral components of chromatin in distant parts of the bacterial kingdom

sample_organism:
Bdellovibrio bacteriovorus
Aquifex aeolicus

overall_design: Bdellovibrio Bacteriovorus : Three independent biological replicates; Aquifex aeolicus: two independent technical replicates

first: Antoine

last: Hocher

first: Shawn

last: Laursen

first: Jess

last: Tyson

first: Carey

last: Lambert

first: Kathryn

last: Stevens

first: Alex

last: Montoya

first: Pavel

last: Shliaha

first: Mathieu

last: Picardeau

first: Elizabeth

last: Sockett

first: Karolin

last: Luger


title: Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina I

last: Tan

overall_design: To investigate circadian regulation of autophagy and lysosomal genes, we collected total RNA from mouse RPE at 8 different timepoints (0, 1, 3, 5, 6, 9, 15, 16 h after light onset). 3 mice (replicates) were included for each timepoint. Light onset is at 7 am.


title: Sex differences in offspring risk and resilience following 11β-hydroxylase antagonism in a rodent model of maternal immune activation

city: Pittsburgh

first: Micah

middle: Aaron

last: Shelton

state: Pennsylvania

address: 2602 PITCAIRN RD

department: Psychiatry

institute: University of Pittsburgh

summary: Maternal immune activation (MIA)puts offspring at greater risk for neurodevelopmental disorders associated with impaired social behavior. While it is known that immune signaling through the maternal, placental, and fetal compartments contribute to these phenotypical changes, it is unknown to what extent the stress response to illness is involved and how it can be harnessed for potential interventions. To this end, pregnant rat dams were treated with the clinically available 11β-hydroxylase inhibitor metyrapone, alongside administration of the bacterial mimetic lipopolysaccharide (LPS), on gestational day 15. Maternal, placental, and fetal CNS levels of corticosterone and placental 11ßHSD enzymes type 1 and 2 were measured 3-hrs post treatment. Subsequently, offspring social behaviors were measured across critical phases of development. MIA was associated with increased maternal, placental, and fetal CNS corticosterone concentrations that were diminished with metyrapone exposure. Metyrapone protected against reductions in placental 11ßHSD2 in males only, suggesting that less corticosterone was inactivated in female placentas. Behaviorally, metyrapone-exposure protected against MIA-induced social disruptions in juvenile, adolescent, and adult males, while females were unaffected or performed worse. Transcriptomic analyses revealed that metyrapone-exposure triggered opposing effects on gene expression to combat MIA-exposure in males, but not among females. Taken together, these findings illustrate that MIA-induced HPA responses act alongside the immune system to produce behavioral deficits.  As a clinical drug already provided to pregnant people and neonates for the treatment of hypercortisolism, the sex-specific benefits and constraints of metyrapone should be investigated further as a potential means of reducing neurodevelopmental risks due to gestational MIA.

overall_design:
To investigate the effect of the maternal stress on the developmental trajectory of male and female rats, pregnant rat dams were treated with the clinically available 11β-hydroxylase inhibitor metyrapone, alongside administration of the bacterial mimetic lipopolysaccharide (LPS), on gestational day 15.
We then isolated RNA from ventral hippocampus of the offspring of treated animals and controls, and performed RNA-seq of total RNA from rats and compared gene expression between groups.

first: Julia

last: Martz

first: Laurel

last: Geist

first: Marianne

last: Seney

first: Amanda

last: Kentner


title: A single-cell atlas of the single versus multiple parous Hu Sheep ovary

city: xianyang

first: ting

last: Ge

address: No. 3 Taicheng Road, Yangling Demonstration Zone, Xianyang City, Shaanxi Province

country: China

institute: College of Animal Science and Technology, Northwest A&F University

summary: Follicular development is a highly coordinated process in Hu sheep. Follicle-cyclic recruitment, spatial displacement, follicle atresia, and ovulation are implicated events resulting from the somatic cells' release of molecular signals. Hu sheep is a high-quality sheep breed with high fecundity in China and is ideal for investigating high reproductive traits. In the current study, the sheep with lambing number ≥3 in three consecutive lambing records were assigned to the HLS group, and lambing number = 1 as the LLS group selected from the same farm with three consecutive lambings. Three randomly picked ewes were slaughtered within 12 h of estrus, and unilateral ovarian tissue was collected and analyzed by single-cell RNA sequencing in each group. A total of five types of somatic cells were identified, and corresponding expression profiles were mapped in the ovaries of the Hu sheep. Additionally, the results of the difference in ovary somatic cell expression profiles between HLS and LLS present that the differences between multiples vs. singleton Hu sheep were mainly clustered in the GCs. In addition, 4 granulosa cell subtypes were identified. GeneSwitches results revealed the opening of JPH1 expression and the closure of LOC101112291, which leads to different evolutionary directions of the granular cells. The expression levels of FTH1 and FTL in GCs of Hu sheep in the HLS group were significantly higher, which inhibited necroptosis and ferroptosis of mural-GCs from decreasing follicular atresia. This study constructed the cellular atlas of the ovary and revealed related biological characteristics at the cellular molecular level. It provides a theoretical basis for the mechanisms underlying the differences in ovulation numbers, which contributes to breeding high-fertility sheep and molecular genetics-based selection.

overall_design: six sample from Hu Sheep ovary tissue

first: Ting

first: Yifan

last: Wen

first: Bo

last: Li

first: Xiaoyu

last: Huang

first: Shaohua

last: Jiang

first: Enping

last: Zhang


title: Germinal centers continually recruit naïve T cells that alter the balance between follicular T helper  and follicular regulatory T cells.

status: Public on Aug 19 2023

city: NEW YORK

first: Thiago

middle: Y

last: Oliveira

state: New York

address: 1230 YORK AVE

department: Immunology, Virology and Microbiology

institute: The Rockefeller University

summary: Follicular helper T cells (T FH ) mediate B cell selection and clonal expansion in germinal centers (GCs), and follicular regulatory T cells (T FR ) prevent the emergence of self-reactive B cells and help to extinguish the reaction. Here we show that GC reactions continually recruit T cells from both the naïve conventional and naive thymic regulatory T cell (Treg) repertoires. In the early GC, newly recruited T cells develop into T FH , whereas cells entering during the contraction phase develop into T FR cells that contribute to GC dissolution. The T FR fate decision is associated with decreased antigen availability and is modulated by slow antigen delivery or mRNA vaccination. Thus, invasion of ongoing GCs by newly developing T FH and T FR from naïve conventional and Treg repertoires, helps remodel the GC based on antigen availability.

overall_design: For single cell RNA sequencing, single cell suspensions were prepared from half-spleens of NP-OVA immunized SellCreERT2 ROSAtdT mice on day 7 and 21 after immunization. Samples were indexed with TotalSeqC (BioLegend) cell surface antibodies and CD4 + , CD62 low , CD44 hi , PD1 hi , CXCR5 high , tdTomato + Tfh cells were purified by flow cytometry, pooled and loaded onto a Chromium Controller (10x Genomics). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10x Genomics) according to manufacturer’s protocol. Libraries were loaded onto an Illumina NextSeq with the mid-Output Kit (150 paired end) for V-D-J analysis or NOVAseq for single cell gene expression. Hashtag indexing was used to demultiplex the sequencing data and generate gene- barcode matrices, respectively.

last: Merkenschlager

first: Riza

middle: M

last: Bernz

first: Victor

last: Ramos

first: Maximilan

last: Uhlig

first: Andrew

last: MacLean

first: Carla

middle: R

last: Nowosad

first: Michel

last: Nussenzweig


title: H3K27ac Hi-ChIP analysis in Jurkat and RPMI-8402 T-ALL cells

first: Tze King

last: Tan

summary: H3K27ac Hi-ChIP analysis was performed in Jurkat and RPMI-8402 stably expressing dCAR9-KRAB after treatment with DMSO (Control) or Doxycycline to induce sgRNA expression to block MYCN enhnacer region

overall_design: Cells were treated with DMSO or doxycycline for 48 hours.  The cells were subsequently collected and  crosslinked with formaldehyde for 10 min.  Hi-ChIP was performed using an antibody against H3K27ac (Abcam: ab4729). Cells were first lysed and then incubated with antibody against H3K27ac histone mark.  DNA was enriched by chromatin-immunoprecipitation (ChIP), tagmented with Tn5, subjected for library construction, and sequenced by the DNBseq platform.

first: Shi

first: Tze

first: Takaomi

last: Sanda


title: ATAC-seq analysis for Jurkat nd RPMI-8402 T-ALL cells

summary: ATAC-seq analysis was performed in Jurkat and RPMI-8402 to analyze chromatin accessibility at steady state

overall_design: The cells were first incubated with Tn5 transposase. Tagmented DNA fragments are purified with Zymo DNA purification kit. ATAC libraries are purified and sequenced on Illumina NovaSeq at GENEWIZ Azenta Life Sciences Co. Ltd.


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [Micro-Capture C]

city: Oxford

first: Thomas

last: Milne

address: John Radcliffe Hospital, Headington

department: MRC Weatherall Institute of Molecular Medicine

institute: University of Oxford

overall_design: Micro-Capture-C

first: Nicholas

last: Crump

first: Alastair

last: Smith

last: Denny

first: Joseph

last: Hamley

first: James

middle: O

last: Davies


title: Loss of STIM1 leads to pancreatic beta cell dysfunction and dedifferentiation in females via GPER downregulation

city: Indianapolis

first: Carmella

last: Evans-Molina

state: Indiana

address: 16 Tech Innovation Bldg 1; 1210 Waterway Blvd Ste 4100

department: Pediatrics

institute: Indiana University School of Medicine

summary: STIM1 is an endoplasmic reticulum (ER) calcium (Ca2+) sensor that serves to replenish ER Ca2+ stores in response to ER Ca2+ depletion through gating of plasmalemmal Orai1 channels in a process known as store operated calcium entry (SOCE). Previously, we have shown that SOCE is impaired in the diabetic pancreatic β cell; however, the consequences of reduced β cell SOCE have not been well studied. To test this, we generated mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ). Our results revealed a striking sexual dimorphism in metabolic phenotypes when STIM1Δβ mice were challenged with high fat diet for 8 weeks. Male STIM1Δβ mice showed no differences in total weight, lean or fat mass, or glucose tolerance compared to WT littermate controls. In contrast, female STIM1Δβ mice displayed significantly increased total body weight, fat mass, and reduced glucose tolerance, in vivo glucose-stimulated insulin secretion and β cell mass compared to WT littermate controls, while insulin sensitivity, food intake, and energy expenditure were unchanged.    To investigate mechanisms underlying these findings, RNA sequencing was performed in isolated islets and revealed altered 17-beta estradiol (E2) signaling, lipid metabolism, epithelial cell differentiation and decreased noncanonical estradiol receptor GPER. Consistent with this, alpha cell mass was increased in female STIM1Δβ, while proteomics and immunoblot analysis of STIM1 knockout (STIM1KO) INS-1 beta cells revealed reduce insulin expression and increased glucagon expression, suggesting STIM1 may be required for the maintenance of β cell identity. To this end, we show that a reduction of a noncanonical estradiol receptor GPER in STIM1 deficient islets contributes to the marked sexual dimorphism observed during beta cell dysfunction in female STIM1Δβ mice. This present study delineates a sexually dimorphic regulation of STIM1 in the maintenance of pancreatic beta cell identity through GPER and may expand the field of therapeutic approaches to diabetes. Our findings demonstrate the importance of STIM1 and GPER expression stability in the anti-diabetogenic response from estradiol.

overall_design: Identifying the molecular changes as a results of pancreatic beta cell specific STIM1 knockout.

first: Paul

last: Sohn

first: Preethi

last: Krishnan

first: Tatsuyoshi

last: Kono

first: Chih-Chun

last: Lee


title: Combination therapies to improve the efficacy of immunotherapy in triple-negative breast cancer [RNA-seq]

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Immune checkpoint inhibitors combined with chemotherapy represent a promising treatment option in triple-negative breast cancer (TNBC). However, response rates are still relatively low necessitating the design of novel therapeutic strategies to improve clinical outcomes. Here, we describe a triple combination of anti-PDL-1 immune checkpoint blockade, epigenetic modulation thorough BET bromodomain inhibition, and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine breast cancer models. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T and B cell infiltration and macrophage reprogramming from M1 to a M2 phenotype in mice treated with triple combination.

overall_design: 5- to 6-week-old BALBc mice were injected with 200,000 EMT-6 mammary tumors cells (ATTC, CRL-2755). Both inguinal mammary fat pads were injected in each mouse. Tumor development was monitored by palpation and once tumors were palpable, usually about 5-7 days after cancer cell inoculation, treatment was initiate.  JQ1 was administered as single agents or in combination at the following doses: JQ1 at 50mg/kg by daily gavage,, anti-PD-L1 antibody (clone 10F.9G2, BioXCell) and paclitaxel each at 10mg/kg by i.p. injection twice per week. Tumor volumes were measured by caliper every three days until the end of the experiment. Once control tumors reached the study endpoint, all mice were euthanized, and primary tumors and various organs were collected. Tumors were then dissociated into single cells and separated into CD45+ and CD45- cell fractions using anti-CD45 antibody coupled to Dynabeads to enrich for leukocytes and tumor cells, respectively. Cells were counted and about 50,000 cells for each fraction were frozen for bulk ATAC-seq. The remaining cells were used for RNA extraction and bulk RNA-seq.

first: Maša

last: Alečković

first: Zheqi

last: Li


title: Reduced FOXF1 Links Unrepaired DNA Damage to  Pulmonary Arterial Hypertension

city: Stanford

first: Sarasa

last: Isobe

address: 269 Campus Drive, CCSR 1220

department: Pediatrics, Cardiology

institute: Stanford University

summary:
Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common mutant gene in PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxic exposure (reoxygenation), as do mice with EC deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension.
Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC revealed reduced Foxf1, a transcription factor with relative selectivity for lung EC.  Reducing FOXF1 in control PAEC induced DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repaired DNA damage and restored angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repaired DNA damage, induced angiogenesis and reversed pulmonary hypertension.

overall_design: Comparative gene expression profiling analysis of RNA-seq data and scRNA-seq for lung endothelial cells from transgenic mice.

first: Ramesh

middle: V

last: Nair

first: Helen

last: Kang

first: Jesse

last: Engreitz

first: Michael

last: Snyder

first: Marlene

last: Rabinovitch


title: Combination therapies to improve the efficacy of immunotherapy in triple-negative breast cancer

overall_design: Refer to individual Series


title: Tissue-resident, extravascular Ly6c- monocytes are critical for inflammation in the synovium

first: Deborah

last: Winter

address: 240 E. Huron Street, M300


title: Combination therapies to improve the efficacy of immunotherapy in triple-negative breast cancer [ATAC-seq]


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [TOPmentation data]

first: Thomas

last: Milne

overall_design: ChIP-seq

first: Nicholas

last: Crump

first: Alastair

last: Smith


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [Capture-C]

overall_design: Next generation Capture-C


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [ChIP-Seq]

first: Ana

last: Dopico-Fernandez


title: Hyperosmotic stress induces 2-cell-like cells through ROS and ATR signaling

city: Munich

first: Maria Elena

last: Torres-Padilla

address: Feodor-Lynen-Strasse 21

department: Institute of Epigenetics and Stem Cells

institute: Helmholtz Zentrum München

summary: Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. ESC cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2-cell embryo, referred to as 2-cell-like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive-oxygen species (ROS) as well as ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic-mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-sequencing at 3 different time point of sorbitol treatment for FACs-sorted ESC based on tbGFP content

first: Antoine

last: Canat

first: Derya

last: Atilla

first: Maria-Elena


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [TOPmentation]


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [ChIP-seq]


title: The lncRNA Sweetheart regulates adaptive cardiac hypertrophy after myocardial injury

city: Frankfurt am Main

first: Phillip

last: Grote

state: Hessen

address: Paul-Ehrlich-Str. 42-44

department: Genome Regulation

institute: Georg-Speyer-Haus

summary: NOT PROVIDED; REQUESTED

overall_design: RNA profile of WT compared to Swhtr mutant heart slices after 7d hypoxia (3% O2) followed by 7d normoxia (21% O2)

first: Sandra

last: Rogala

first: Tamer

last: Ali


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [RNA-seq]

overall_design: RNA-seq


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [TT-Seq]


title: Regulation of transpiration hinges on spatial differences in abscisic acid perception

first: Idan

last: Efroni

address: POB 12

department: Plant Sciences

institute: Hebrew University

summary: The plant hormone abscisic acid (ABA) is best known for its role as a regulator of the responses to abiotic stressors. Components of ABA signaling pathway are thus considered as promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting a number of physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors called PYR/PYL/RCAR. Arabidopsis genome encodes 14 PYL proteins, knowledge on differential biological functions of these members is scarce. In this work, we took a gain-of-function approach to predict receptor-specific functionality. We introduced a set of activating mutations in the mobile gate and α-helix of sub family II ABA receptors. These mutations constitutively enforce the active ABA-bound conformation. We then transformed ABA deficient mutants with the constitutive receptors and monitored suppression of ABA deficiency phenotype. Our findings suggest that subfamily II receptors have differential activity in regulating transpiration and transcription of oxidation and stress response genes. These differences partly derived from differential accumulation of PYLs in the guard cells. Data mining revealed that only a small subset of PYL receptors are transcribed in the guard cells in a steady state. Because the guard cell specific receptors have different affinity to ABA and to the PP2C co-receptor we propose that, a combined partial receptor redundancy facilitate a gradual activation of the ABA output signal as its cellular concentration rises via biochemically different receptors.

overall_design: Whole PYL/PYR seedlings mock treated, and whole Col-0 and aba2-1 seedlings mock treated or treated with 10nM ABA or 50nM ABA.

first: Assaf

last: Mosquna

first: Oded

last: Pri-Tal

first: Jan

last: de Vries

first: Janine

last: Fürst-Jansenc

first: Armin

last: Dedras


title: Cyclo (His-Pro): a further step in the prevention of steatohepatitis

status: Public on Aug 14 2023

city: Lausanne

first: Keno

last: Strotjohann

address: Route Cantonale

department: School of Life Sciences, Institute of Bioengineering

country: Switzerland

institute: École Polytechnique Fédérale de Lausanne (EPFL)

summary: Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the world, and despite this has no approved therapy. The naturally occurring compound Cyclo (His-Pro) (CHP) has been described as having anti-inflammatory and hypoglycemic properties. We show that CHP administration in a mouse model of NAFLD/NASH protects against disease progression. Based on our study, CHP prevents overall lipid accumulation, reducing body weight and fat mass. The treatment lowers systemic inflammation and prevents the development of insulin resistance. Specifically, the effect of CHP in the liver is remarkable, it reduces the steatosis while preventing inflammation and fibrosis, typical features of NADLF/NASH progression. The analysis of the liver transcriptome underscores the anti-fibrotic and anti-inflammatory effects of the treatment, supported by histology results. CHP, which has an excellent safety record, may hence represent a novel approach to manage NAFLD.

overall_design: We assessed the effectiveness of CHP on the evolution of NAFLD/NASH that was induced in male C57BL/6J mice by housing them at thermoneutrality (TN) and feeding a western diet (WD). Histological and molecular characterizations were performed to assess the validity of our NASH mouse model and the effect of CHP in the context of liver disease.

first: Alessia

last: De Masi

first: Xiaoxu

last: Li

first: Qi

last: Wang

first: Hoe-Yune

last: Jung

first: Johan

last: Auwerx


title: Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment II

city: Tokyo

first: Tomohiro

last: Yabushita

address: 7-3-1, Hongou, Bunkyo-ku

country: Japan

institute: The University of Tokyo

summary: Decitabine (DAC) is used clinically for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells shows that mitotic regulation plays a pivotal role in DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations and antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, the overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation, while highlighting the potent activity of DAC to perturb mitosis through aberrant DNMT1-DNA covalent bonds.

overall_design: To identify genes that regulate sensitivity and resistance of AML cells to DAC and SAHA, we performed a genome-wide pooled CRISPR activation screen using cSAM (engineered murine sAML (MDS/AML) cells transformed by combined expression of SETBP1 and ASXL1 Mutations cells expressing the Suntag system.

first: Toshio

last: Kitamura

first: Susumu

last: Goyama


title: Photoperiodic Transcriptome of Arabidopsis thaliana

city: New Haven

first: Chun Chung

last: Leung

state: CT

address: YSB 424, 260 Whitney Avenue

department: MCDB

institute: Yale University

summary: Photoperiod is a circannual signal measured by biological systems to align growth and reproduction with the seasons. To understand the effect of photoperiod of gene expression in Arabidopsis thaliana in the absence of exogenous sugar under constant light intensity, we performed time course mRNA-seq analysis on 13-day old seedlings across three photoperiods with triplicates to identify photoperiod-regulated genes.

overall_design: Time course bulk mRNA seq analysis of Arabidopsis seedling gene expression across three photoperiods: long day (16L:8D), equinox (12L:12D) and short day (8L:16D). Seedlings were grown without exogenous sugar for 10 days in equinox photoperiod before transfer to the respective photoperiod for 2 days. Whole seedlings were collected every four hours at Zeitgeber time 00 h, 04 h, 08 h, 12 h, 16 h and 20 h.

first: Chun

first: Joshua

last: Gendron


title: Single-cell epigenome analysis identifies transcriptional events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells [ATAC-seq]

city: Helsinki

first: Biswajyoti

last: Sahu

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary: Cell fate can be reprogrammed by ectopic expression of lineage-specific transcription factors (TF). However, the exact cell state transitions during transdifferentiation are still poorly understood. Here, we have generated pancreatic exocrine cells of ductal epithelial identity from human fibroblasts using a set of six TFs. We mapped the molecular determinants of lineage dynamics using a factor-indexing method based on single-nuclei multiome sequencing (FI-snMultiome-seq) that enables dissecting the role of each individual TF and pool of TFs in cell fate conversion. We show that transition from mesenchymal fibroblast identity to epithelial pancreatic exocrine fate involves two deterministic steps: an endodermal progenitor state defined by activation of HHEX with FOXA2 and SOX17, and a temporal GATA4 activation essential for maintenance of pancreatic cell fate program. Collectively, our data suggest that transdifferentiation – although being considered a direct cell fate conversion method – occurs through transient progenitor states orchestrated by stepwise activation of distinct TFs.

overall_design: Chromatin accessibility was analyzed by ATAC-seq in control human foreskin fibroblasts (HFFs) and reprogramming cells.

first: Liangru

last: Fei

first: Kaiyang

last: Zhang

first: Nikita

last: Poddar

first: Sampsa

last: Hautaniemi


title: Single Cell Transcriptomic Analysis of Popliteal Lymph Nodes from TNF-Tg Mice with Early and Advanced Inflammatory-Erosive Arthritis

city: Rochester

first: H. Mark

last: Kenney

address: 601 Elmwood Ave, Box 665

department: Center for Musculoskeletal Research

institute: University of Rochester Medical Center

summary: Immune cell dynamics in lymph nodes downstream of inflamed joints are associated with inflammatory-erosive arthritis in both tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. In TNF-Tg mice, "Early" stages of arthritis are related to dramatic PLN "Expansion" with increases in joint-draining popliteal lymph node (PLN) volume and fluid flow, while CD21-high / CD23+ B-cells in inflamed nodes (Bin cells) accumulate in the PLN follicles. Onset of "Advanced" inflammatory-erosive arthritis is related to a PLN "Collapse" where fluid flow is reduced as Bin cells translocate from the PLN follicles into the sinuses and are thought to mechanically inhibit lymphatic drainage. Through single-cell RNA-sequencing, we aimed to evaluate changes in PLN immune cell populations during Early and Advanced arthritis to determine mechanisms mediating the Bin cell translocation and the relationship with arthritic progession in the afferent joints.

overall_design: Two total single-cell RNA-seq samples are provided (n=3 mice, 6 PLNs pooled per sample); both samples are from TNF-Tg male mice, one with Early arthritis (5-6 months of age) and one with Advanced arthritis (>8 months of age). TNF-Tg females exhibit early mortality related to cardiopulmonary disease, and thus male mice are utilized to investigate these chronic lymphatic processes. The TNF-Tg mice are on a C57BL/6 genetic background.

first: Edward

last: Schwarz

first: H

first: Chia-Lung

last: Wu


title: Cyclo(His-Pro): A further step in the management of steatohepatitis

first: Xiaoxu

last: Li

address: Station 15 - CH

institute: EPFL

summary: Non-alcoholic fatty liver disease (NAFLD) is now considered the most common liver disease in the world, yet no pharmacological treatment has been approved. The naturally occurring compound Cyclo (His-Pro) (CHP) appears as an interesting candidate for the management of NAFLD, given its safety records and its anti-inflammatory effect. We show here that CHP administration protects against progression towards steatohepatitis (NASH) and fibrosis in two different mouse models of liver injury: a model of dietary NAFLD/NASH, achieved with thermoneutral housing in combination with a western diet, and a model of liver fibrosis by repeated injections with carbon tetrachloride. CHP administration prevents overall lipid accumulation, reducing body weight and fat mass. Treated animals show lower systemic inflammation and improved glycemia. Histo-pathology and liver transcriptomics highlight reduced steatosis upon CHP, together with lower inflammation and fibrosis, which are typical features of NAFLD progression. For the first time, we identified ERK as an early mediator of CHP response, although more work is required to elucidate its precise mechanism of action. In conclusion, our work indicates that CHP is a novel and efficacious strategy to manage NAFLD, fuelling optimism for potential clinical studies.

overall_design: A model of acute liver injury was then used. C57BL/6N mice received repeated low-dose (0.2ml/kg) CCl4 injections (CCL4:Olive oil = 1:19) over two weeks, together with daily CHP treatment (35mg/kg by oral gavage)

first: Alessia

last: De Masi

first: Qi

first: Hoe-Yune


title: Single-cell epigenome analysis identifies transcriptional events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells [RNA-seq]

overall_design: Gene expression profiling by RNA-seq in control human foreskin fibroblasts (HFFs) and reprogramming cells.


title: Single-cell epigenome analysis identifies transcriptional events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells [ChIP-seq]

overall_design: Enrichment of histone marker H3K27ac in control human foreskin fibroblasts (HFFs) and reprogramming cells was analyzed by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq).


title: MLL-AF4 cooperates with PAF1 and FACT to drive high density enhancer interactions in leukemia [ATAC-seq]

first: Thomas

last: Milne

summary: Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT and loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia, but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.

overall_design: ATAC-seq

first: Nicholas

last: Crump

first: Alastair

last: Smith


last: Wang

last: Jung

first: Johan

last: Auwerx


title: Single-cell epigenome analysis identifies transcriptional events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells [FI-snMultiome-seq]

first: Biswajyoti

summary: Cell fate can be reprogrammed by ectopic expression of lineage-specific transcription factors (TF). However, the exact cell state transitions during transdifferentiation are still poorly understood. Here, we have generated pancreatic exocrine cells of ductal epithelial identity from human fibroblasts using a set of six TFs. We mapped the molecular determinants of lineage dynamics using a factor-indexing method based on single-nuclei multiome sequencing (FI-snMultiome-seq) that enables dissecting the role of each individual TF and pool of TFs in cell fate conversion. We show that transition from mesenchymal fibroblast identity to epithelial pancreatic exocrine fate involves two deterministic steps: an endodermal progenitor state defined by activation of HHEX with FOXA2 and SOX17, and a temporal GATA4 activation essential for maintenance of pancreatic cell fate program. Collectively, our data suggest that transdifferentiation – although being considered a direct cell fate conversion method – occurs through transient progenitor states orchestrated by stepwise activation of distinct TFs

overall_design: Gene expression and chromatin accessibility from the same cell was measured by single-nuclei (sn) RNA-seq & ATAC-seq in control human foreskin fibroblasts (HFFs) and reprogramming cells.


title: Spatial Transcriptomics of Joint-Draining Popliteal Lymph Nodes from Wild-type and TNF-Tg Mice with Early and Advanced Inflammatory-Erosive Arthritis

summary: Lymphatic dysfunction is an integral aspect of rheumatoid arthritis (RA) pathogenesis. During "Early" arthritis in tumor necrosis factor transgenic (TNF-Tg) mice, the joint-draining popliteal lymph nodes (PLNs) dramatically expand in volume. After a prolonged expansion phase, at ~8-months-old the PLNs stochastically collapse with reduced bone volume and associated onset of "Advanced" inflammatory-erosive arthritis in affected joints. Through this pathologic process, B-cells are known to translocate into the PLN sinuses. Bulk tissue approaches (i.e. bulk genomics, flow cytometry, and ex vivo cell cultures) have been insufficient to specifically evaluate the transcriptional changes of B-cells within the sinuses of TNF-Tg PLNs. Thus, we utilized spatial transcriptomics technology to evaluate transcriptional changes in sinus B-cells and the functional genomic relationships with arthritic severity.

overall_design: A single Gene Expression Slide (10X Genomics) was used to evaluate PLNs for spatial transcriptomics with 3 different groups represented on the 4 capture areas: 1) 5.5-month-old wild-type (n = 5 mice, 10 PLNs), 2) 5.5-month-old TNF-Tg with Early arthritis (n = 3 mice, 6 PLNs), and 8.5-9-month-old TNF-Tg with Advanced arthritis (2 replicates with n = 3 mice, 6 PLNs per replicate). Only male mice were used for the study due to early mortality of TNF-Tg females, and all mice were on a C57BL/6 genetic background. Standard protocols from the Visium Methanol Fixation and H&E Staining Guide and Visium Spatial Gene Expression Kit (10X Genomics) were performed.

first: Chia


title: Single-cell epigenome analysis identifies transcriptional events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells

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

overall_design: Refer to individual Series


title: Single-cell multi-omics defines the cell-type specific impact of splicing aberrations in human hematopoietic clonal outgrowths

city: New York

first: Dan

middle: A.

last: Landau

state: NY

address: 101 6th Ave

department: Molecular Pharmacology Program

institute: New York Genome Center

summary: RNA splicing factors are recurrently affected by alteration-of-function mutations in clonal blood disorders, highlighting the importance of splicing regulation in hematopoiesis. However, our understanding of the impact of dysregulated RNA splicing has been hampered by the inability to distinguish mutant and wildtype cells in primary patient samples, the cell-type complexity of the hematopoietic system, and the sparse coverage of splice junctions by short-read sequencing typically used in single-cell RNA sequencing. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT) with enhanced efficiency long-read single-cell transcriptome profiling, as well as proteogenomics (with CITE-seq). This allowed for the simultaneous single-cell profiling of gene expression, cell surface protein markers, somatic mutation status, and RNA splicing. We applied GoT-Splice to bone marrow progenitors from patients with myelodysplastic syndrome (MDS) affected by mutations in the most prevalent mutated RNA splicing factor – the core RNA splicing factor SF3B1. High-resolution mapping of SF3B1mut vs. SF3B1wt hematopoietic progenitors revealed increased fitness advantage of SF3B1mut cells in the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1mut erythroid progenitor (EP) cells. SF3B1mut EP cells exhibited upregulation of genes involved in regulation of cell cycle and mRNA translation. Long-read single-cell transcriptomes revealed the previously reported increase of aberrant 3’ splicing site usage in SF3B1mut cells. However, the ability to profile splicing within individual cell populations uncovered distinct cryptic 3’ splice site usage across different progenitor populations, as well as stage-specific aberrant splicing during erythroid maturation. Lastly, as splice factor mutations occur in clonal hematopoiesis (CH) with increased risk of neoplastic transformation, we applied GoT-Splice to CH samples. These data revealed that the erythroid lineage bias, as well as cell-type specific cryptic 3’ splice site usage in SF3B1mut cells, precede overt MDS. Collectively, we present an expanded multi-omics single-cell toolkit to define the cell-type specific impact of somatic mutations on RNA splicing, from the earliest phases of clonal outgrowths to overt neoplasia, directly in human samples.

overall_design:
We profiled CD34+ enriched cells from GCSF mobilized bone marrow/stem cell graft samples (n = 11) using  single-cell RNA sequencing (10X and ONT) with targeted genotyping and ADT profiling of cell surface markers (for 2 samples, one with 2 replicates).
Submitter states: The dataset contains human patient samples which are on submission process in EGA.

first: Federico

last: Gaiti

first: Paulina

last: Chamely

first: Allegra

middle: G

last: Hawkins

first: Mariela

last: Cortés-López

first: Robert

middle: F

last: Stanley

first: Ariel

middle: D

last: Swett

first: Saravanan

last: Ganesan

first: Tarek

last: Mouhieddine

first: Kiran

last: Batta

first: Nili

last: Furer

first: Rahul

last: Vedula

first: Ronan

last: Chaligné

first: Daniel

last: Wiseman

first: Amos

last: Tanay

first: Liran

last: Shlush

last: Lindsley

first: Irene

last: Ghobrial

first: Justin

last: Taylor

first: Omar

last: Abdel-Wahab


title: A rationally designed dual PI3-kinase (PI3K) and BET-inhibitory chimeric small molecule demonstrates potent anti-neoplastic activity versus MYC-driven haematological cancers in vivo [RNA-seq]

city: South San Francisco

first: Simon

middle: J

last: Hogg

state: California

address: 1000 Gateway Boulevard

department: Oncology Discovery Research

institute: AbbVie

summary: We hypothesised that PI3K and BET inhibition would synergise in the context of MYC-dysregulation and overcome resistance to single-target inhibition. We then synthesised a series of chimeric molecules linking BET- and PI3K-selective pharmacophores, including our lead molecule, 18D(S). RNA-Seq profiling following acute 18D(S) treatment demonstrated robust transcriptional modulation with concomitant BET and PI3K inhibitory signatures. Our data provides proof-of-concept for the design of potent PI3K- and BET-inhibitors, providing ‘synergy in a single molecule’.

overall_design: Acute treatment of OPM2 cells with JQ1, PI3K inhibitor, and 18DS, or DMSO vehicle, prior to collection of RNA for RNA-sequencing to determine differential gene expression changes.

first: Danielle

last: Oh

first: Jake

last: Shortt


title: PTEN regulates chromatin accessibility and hematopoietic lineage plasticity via the transcription factor PU.1

city: Beijing

first: Zihan

last: Xu

address: No.5 Yiheyuan Road Haidian District

institute: Peking University

summary: We elucidate the molecular and cellular mechanism underlying PTEN-regulated hematopoietic lineage choice and suggest a critical role of PU.1 in modulating the epigenetic state of prepro-B progenitors.

overall_design: Examination of chromatin accessibility in stem and progenitor cells from WT and PTEN loss mouse bone marrow. Examination of chromatin accessibility and expression level in prepro-B cells from WT and PTEN loss mouse bone marrow.

first: Yilin

last: Wu


title: Transcriptome analysis reveals key genes and pathways that macrophage of islet in NCD(normal chow feed) or HFD( high fat feed)

city: Jinan

first: Jin-Peng

last: Sun

address: 44 Wenhua Xi Rd

institute: Shandong University

summary: Our research is helpful to understand the physiological and pathological level changes caused on the macrophages of islet in HFD conditional, to find the different mechanism or signaling pathway compared with NCD

overall_design: In this study, we isolated macrophages in islet from WT mice fed NCD or HFD. Every group (2 samples each) were subjected to RNA sequencing.

first: J

last: Cheng


title: Mouse genome rewriting and tailoring of three important disease loci [RNA]

status: Public on Aug 13 2023

first: Weimin

last: Zhang

address: 435 E 30th street

department: Institute for Systems Genetics

institute: NYU Langone Health

summary: Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection. Lastly, we demonstrated serial genome writing by humanizing mouse Tmprss2 in a biallelic fashion, highlighting the versatility of mSwAP-In in mouse genome writing.

overall_design: For the SARS-CoV-2 infected mouse lungs, three groups (WT, K18-ACE2, hACE2) of lungs were collected. Each group contains two infected animals with 10^5 PFU SARS-CoV-2, and two uninfected control animals. For the doxorubicin treated mESCs, two groups (WT, synTrp53) of mESCs were harvested, each group contains two 250 nM 20h doxorubicin treated clones, and two untreated control clones.

first: Jef

last: Boeke


title: Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice

city: Los Angeles

first: Yuichiro

last: Itoh

address: 635 Charles E. Young Drive South

department: Neurology

institute: UCLA

summary: Menopause is associated with cognitive deficits and brain atrophy, but the brain region and cell-specific mechanisms are not fully understood. Here, we identify a sex hormone by age interaction whereby loss of ovarian hormones in female mice at midlife, but not young age, induced hippocampal-dependent cognitive impairment, dorsal hippocampal atrophy, and astrocyte and microglia activation with synaptic loss. Selective deletion of estrogen receptor beta (ERβ) in astrocytes, but not neurons, in gonadally intact female mice induced the same brain effects. RNA sequencing and pathway analyses of gene expression in hippocampal astrocytes from midlife female astrocyte-ERβ conditional knock out (cKO) mice revealed Gluconeogenesis I and Glycolysis I as the most differentially expressed pathways. Enolase 1 gene expression was increased in hippocampi from both astrocyte-ERβ cKO female mice at midlife and from  postmenopausal women. Gain of function studies showed that ERβ ligand treatment of midlife female mice reversed dorsal hippocampal neuropathology.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hippocampus astrocyte RNAs

first: Noriko

first: Rhonda

last: Voskuhl


title: Mouse genome rewriting and tailoring of three important disease loci [ChIP]

overall_design: Three groups (WT, 116kb-hACE2, 180kb-hACE2) of mouse testes were included in this dataset, each group has two biological replicates, being pulled down with IgG negative control, and H3K4me3 and H3K27ac antibodies.


title: ERO1A drives immunosuppression and attenuates response to immune checkpoint blockade

city: Chengdu, Cihna

first: Xaingyu

last: Pan

state: Sichuan

address: No.17 Renming road section3

department: State Key Laboratory of Biotherapy, West China Hospital, Sihcuan University.

institute: Sichuan University

summary: Immunophenotyping of tumor microenvironment (TME) is crucial for immunotherapy efficacy in patients with solid tumours. However, strategies to characterize TME are largely limited with huge heterogeneity. We show that endoplasmic reticular oxidoreductase-1α (ERO1A) induces an immune-suppressive TME and resistance to PD-1 blockade by promoting endoplasmic reticulum (ER) stress response. Single-cell RNA sequencing analyses confirm that ERO1A is correlated with immunosuppression and dysfunction of CD8+ T cell along anti-PD-1 treatment. Ablation of Ero1a in tumours promotes the infiltration of lymphocytes as well as cytotoxicity of CD8+ T cells and enhances response to anti-PD-1 treatment in mouse models.

overall_design: To dissect the cellular and molecular changes of the TME during immunotherapy, we performed scRNA-seq analyses of tumours from wild type mice (n=5) and Ero1a KO mice (n=5).

first: Lihui

last: Liu

first: Xiangyu

first: Jian

last: Wang


title: The LysR‐type transcription factor LsrB regulates beta‐lactam resistance in Agrobacterium tumefaciens

status: Public on Aug 12 2023

city: Bochum

first: Franz

last: Narberhaus

state: NRW

address: Universitätstraße 150

department: Microbial Biology

institute: Ruhr University Bochum

summary: We investigated the role of the LysR-type transcriptional regulator LsrB (Atu2186) from Agrobacterium tumefaciens C58 (alias A. fabrum C58), which regulates the expression of small-regulatory-RNA (sRNA) and several protein-coding genes, such as ampD (atu2113). In our current study we invesigated the role of LsrB in beta-lactam resistance. For this purpose, we constructed a vtlR/lsrB deletion mutant. Total RNA was isolated from this mutant. Sequencing was done on the Illumina NovaSeq 6000 platform. Wild-type samples that derived from the same experiment were published previously (Kraus et al., 2020) (GEO accession: GSE150941). In the vtlR/lsrB mutant around 800 protein coding genes and around 80 sRNA coding genes were downregulated or  upregulated at least three-fold (p value ≤ 0.05). Overall, the results demonstrate the huge regulatory effect of LsrB on the  transcriptome of A. tumefaciens.    Kraus, A., Weskamp, M., Zierles, J., Balzer, M., Busch, R., Eisfeld, J., et al. (2020) Arginine-rich small proteins with a domain of unknown function DUF1127 play a role in phosphate and carbon metabolism of Agrobacterium tumefaciens. J Bacteriol 202: e00309-20.

sample_organism:
Agrobacterium fabrum str. C58
Agrobacterium fabrum

overall_design:
Illumina RNA sequencing of the transcriptomes of Agrobacterium fabrum C58 wild type, a vtlR/lsrB deletion mutant (∆atu2186).
Re-analysed Samples:
GSM4561620 SAMN14984052 SRX8370442
GSM4561621 SAMN14984051 SRX8370443
GSM4561622 SAMN14984050 SRX8370444

first: Janka

last: Schmidt

first: Donata

last: Remme

first: Jessica

last: Eisfeld

first: Vivian

middle: B

last: Brandenburg

first: Hannah

last: Bille

first: Farnz


title: CXCR6 orchestrates brain CD8+ T cell residency and limits mouse Alzheimer’s disease pathology [scRNA-seq3]

city: Memphis

first: Hongbo

last: Chi

state: TN

address: 262 Danny Thomas Place

department: Immunology

institute: St Jude Children's Research Hospital

summary: Our study reveals a previously unappreciated role for microenvironment-specific, inter-cellular crosstalk between CD8+ T cells and microglia in delaying neurodegenerative disease development

overall_design: We used scRNA-seq to profile CD45+ and CD45- non-immune cells from the brain parenchyma of 5xFAD and 5xFAD;Tcra-/- mice

first: Isabel

last: Risch

first: Hao

last: Shi


title: Identifying biomarkers of heterogeneity and transplantation efficacy in retinal pigment epithelial cells

city: Rensselaer

first: Farhad

last: Farjood

address: 1 Discovery Drive

institute: Neural Stem Cell Institute

summary: Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.


title: Context-dependent Regulation of Notch Signaling in Glial Development and Tumorigenesis [scRNA-seq]

city: Shanghai

first: Zhejun

last: Xu

address: Room B4-029, 131 Dong'an Road, Xuhui

institute: Fudan University

summary: In the mammalian brain, Notch signaling maintains the cortical stem cell pool and regulates the glial cell fate choice and differentiation. However, the function of Notch in regulating glial development and its involvement in tumorigenesis have not been well understood. Here, we show that Notch inactivation by genetic deletion of Rbpj in stem cells decreases astrocytes but increases oligodendrocytes with altered internal states. Interestingly, inhibiting Notch in glial progenitors does not affect cell generation but instead accelerates the growth of Notch-deprived oligodendrocyte progenitor cells (OPCs), and OPC-related glioma. We also identified a crosstalk between oligodendrocytes and astrocytes, with premyelinating oligodendrocytes secreting Bmp4, which is repressed by Notch, to upregulate GFAP expression in adjacent astrocytes. Moreover, Notch inactivation in stem cells causes a glioma subtype shift from astroglia-associated to OPC-correlated patterns, and vice versa. Our study reveals Notch's context-dependent function, promoting astrocytes and astroglia-associated glioma in stem cells and repressing OPCs and related glioma in glial progenitors.

overall_design: scRNA-seq: Individual cells were sorted from the P1 cortices of RbpjF/F; H2B-GFPF/+ mice performed in utero electroporation with pCAG-Cre plasmid at E15.5.

first: Rongliang

last: Guo

first: Danyu

last: Han

first: Xingrui

last: Song

first: Zhengang

last: Yang


title: Context-dependent Regulation of Notch Signaling in Glial Development and Tumorigenesis [Bulk RNA-seq]

overall_design: Bulk RNA-seq: Mice cortices were sorted and collected at P2 from both control (Olig2-Cre; H2B-GFPF/+) and Rbpj-cko (Olig2-Cre; RbpjF/F; H2B-GFPF/+) mice.


title: A rationally designed dual PI3-kinase (PI3K) and BET-inhibitory chimeric small molecule demonstrates potent anti-neoplastic activity versus MYC-driven haematological cancers in vivo [ChIP-seq]

first: Simon

last: Hogg

overall_design: Acute treatment of OPM2 cells with JQ1, PI3K inhibitor, and 18DS, or DMSO vehicle, prior to chromatin immunoprecipitation and sequencing (ChIP-seq) using antibodies for BRD4, H3K27ac, or RNA Polymerase II.

first: Danielle

last: Oh

first: Jake

last: Shortt


title: Mouse genome rewriting and tailoring of three important disease loci

first: Weimin

last: Zhang

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Effects of AR-42 Treatment on Gene Expression in the Zebrafish Tail Fin Amputation (TFA) Model

status: Public on Aug 11 2023

city: Chongqing

first: Sijia

last: Fan

address: Chongqing Medical University, Medical College Road, Yuzhong District

institute: Chongqing medical university

summary: We investigated the inflammatory environmental effects of AR-42 on neutrophil recruitment by performing an RNA-seq analysis with tail tissue in the zebrafish TFA model. The RNA-seq results showed that AR-42 modulates cytokine/chemokine signaling in the local inflammatory environment.

overall_design: For RNA sequencing (RNA-seq) analysis, the 3-dpf WT zebrafish larvae receiving DMSO or 10 μM AR-42 treatments were categorized into 4 groups: ① DMSO treated larvae without TFA modeling (referred to as DMSO), ② AR-42 treated larvae without TFA modeling (AR-42), ③ DMSO treated larvae with TFA modeling (DMSO_TFA) and ④ AR-42 treated larvae with TFA modeling (AR-42_TFA). And there are four biological replicates in each group. After tail fin amputation, larvae were immediately treated with DMSO or 10 μM AR-42 for 1 hr before sampling. Tail tissues posterior to the cloaca in each larva were collected (n = 150) and immediately froze in liquid nitrogen before RNA isolation.

first: Chunguang

last: Ren

first: Jinlong

last: Jiang


title: HD2B and HD2C interact with Argonaute 4 to promote tolerance and heterochromatin stabilization during heat stress [RNA-Seq]

city: Wuhan

first: Xiangsong

last: Chen

state: Hubei

address: Bayi Road

institute: Wuhan university

summary: In this study, we show that HD2B/C work with AGO4 to promote heat tolerance

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and hd2b/c plants under normal and heat-treated conditions,whole-genome bisulfite sequencing in WT and hd2b/c under both normal and heat conditions,high-throughput small RNA sequencing in WT and hd2b/c

first: Yingnan

last: Sun

first: Fangfang


title: Mouse genome rewriting and tailoring of three important disease loci [DNA]

overall_design: Two types of samples are included: 1. Assemblon DNAs or Repository BACs extracted from bacteria. 2. Captured genomic DNAs from engineered mESCs containing mSwAP-In delivered mouse or human DNAs.

first: Jef

last: Boeke


title: Mouse genome rewriting and tailoring of three important disease loci [ATAC]

overall_design: Three groups (WT, 116kb-hACE2, 180kb-hACE2) of mouse small intestines were included in this dataset, each ACE2 humanized mouse group has two biological replicates.


title: Context-dependent Regulation of Notch Signaling in Glial Development and Tumorigenesis

overall_design: Refer to individual Series


title: Circadian modulation by time-restricted feeding restores brain transcription and slows amyloid deposition in a mouse model of Alzheimer’s disease

city: El Cajon

first: Daniel

last: Carlin

address: 1250 Mountain View Rd.

institute: UC-San Diego

summary: Alzheimer’s disease (AD) is a tragic neurodegenerative disease affecting more than 5 million Americans. Circadian disruptions impact nearly all AD patients, with reversal of sleep/wake cycles and agitation in the evening being common disturbances that manifest early in disease. These alterations support a role for circadian dysfunction as a driver of AD, emphasizing a critical need to investigate the therapeutic potential of circadian-modulating interventions. One of the most powerful regulators of the circadian system is the daily feed/fast cycle. Here we show that time-restricted feeding (TRF) without caloric restriction, improved key disease components including behavior, disease pathology, hippocampal transcription, and memory in two transgenic mouse models of Alzheimer’s disease. We found that TRF had the remarkable capability of simultaneously reducing amyloid deposition, increasing Aβ42 clearance, improving sleep and memory, and normalizing time-of-day transcription of multiple genes, including those associated with AD and neuroinflammation. Thus, our study unveils for the first time the multifactorial effects of timed feeding, which has far-reaching impact beyond metabolism and affects the brain as the substrate for neurodegeneration, including the alignment of circadian rhythmicity. Since the pleiotropic effects of TRF can substantially modify disease trajectory, this intervention has immediate translational value, addressing the crucial need for accessible approaches to reduce or halt AD progression.

overall_design: Prior to any measurements or beginning any experiments, mice were habituated to a 12:12 light-dark (LD 12:12) cycle and single housing conditions in custom light-tight cabinets. We then measured diurnal rhythms in activity and sleep behavior from the mice. To assess circadian impairments in the mice, some animals were placed in constant darkness (DD), followed by light masking and 6 h phase advance protocols. Feeding manipulations were then performed on mice habituated to LD 12:12. Animals within each sex and genotype were randomly divided into two groups with one group continuing ALF and the other placed on TRF, with food available during 6 h in the middle of the mouse active phase, during zeitgeber time (ZT) 15 to ZT21. By definition, ZT0 is the time when lights go on and ZT12 is the time when the lights go off when the mice are in a 12:12 LD cycle. After habituating to the feeding protocol, metabolites were measured in the mice to ensure the treatment was inducing the expected metabolic effects. The mice remained on TRF or ALF food access until sacrificed for tissue collection. After all tests were completed and the mice had re-entrained to the LD cycle, mice within each genotype and treatment group were randomly assigned for sacrifice at one of four time points: ZT0, ZT6, ZT12, or ZT18. ZT0 and ZT6 tissues were collected in the light; ZT12 and ZT18 were collected in the dark. Brain tissue and blood serum were collected for analysis. This study used a total of 110 mice almost equally distributed across sex/ genotype/treatment throughout the study, and further grouped by ZT collection times. Treated mice started TRF at6 to 9 months of age and were maintained on TRF until tissue collection, which occurred at 10 to 13 months of age.

middle: S

last: Whittaker

first: Laila

last: Akhmetova

first: Haylie

last: Romero

first: David

middle: K

last: Welsh

first: Christopher

last: Colwel

first: Paula

last: Desplats


title: Cut and Run from Caki-1, MCF7, and U251 cell lines with NRF1 and CTCF

city: Saint Louis

first: Li

last: Ding

state: MO

address: 660 S. Euclid Avenue

institute: Washington University in St. Louis

summary: We performed a Cut & Run assay in the associated cell lines to profile the direct binding of NRF1 and CTCF  at the promoters of target genes.

overall_design: Caki-1, MCF7, and U251 cell lines were cultured under designated conditions according to information on the American Type Culture Collection (ATCC) website (https://www.atcc.org/). When cells reached the desired confluence and numbers, the CUT&RUN Kit (14-1048, EpiCypher, USA) and CUT&RUN Library Prep Kit (14-1002, EpiCyher, USA) were applied following the manufacturer's protocols. Briefly, wash buffer, cell permeabilization buffer, and antibody buffer were freshly prepared on day 1. ConA Beads were activated by washing and then diluted with a cold bead activation buffer. Following these steps, 500,000 cells were harvested for each reaction, followed by resuspending in a wash buffer and mixing well with activated beads. After 10 minutes of incubation at room temperature, tubes were placed on an 8-strip magnet until slurries cleared. Supernatant was removed and a cold antibody buffer was added to each reaction. The SNAP-CUTANATMK-MetStat Panel was first added to the reactions designed for positive (H3K4me3) and negative (IgG) control antibodies. Then, 0.5 ug designated antibody was added to each reaction and incubated overnight. On the next day, antibody-bound histone PTM or chromatin-interacting protein were washed with the cell permeabilization buffer. Next, pAG-MNase was added to cleave target-DNA complexes. Targeted chromatins were then digested and released by adding calcium chloride, E. coli spike-in DNA, and Stop Buffer Master Mix. DNA was purified and up to 5ng CUT&RUN-enriched DNA was used for further library construction following the CUTANATM CUT&RUN Library Prep Kit. Library fragment sizes were analyzed using TapeStation and libraries were sequenced.

first: Jayasinghe

last: Reyka


title: Therapy-induced senescence of cancer-associated fibroblasts determines survival in rectal cancer

city: Frankfurt am Main

first: Florian

last: Greten

address: Paul-Ehrlich-Str. 42-44

department: Institute for Tumor Biology and Experimental Therapy

institute: Georg-Speyer-Haus

summary: Standard therapy of rectal cancer comprises a combination of local radio- and systemic chemotherapy prior to surgery. Despite comparable tumor morphology and stage the outcome of such neoadjuvant therapy is heterogeneous and the underlying factors that define therapy response are unknown. Here we demonstrate that cancer-associated fibroblasts (CAFs) rather than tumor cells themselves have major impact on therapy response and survival in a novel pre-clinical in vivo model of rectal cancer as well as in patients. We show that irradiation of tumors increases DNA damage further in inflammatory CAFs causing them to undergo a p53-dependent senescence program, which is associated with the secretion of various extracellular matrix components that strongly promote therapy resistance and progression. Administration of  venetoclax (senolytics) renders therapy-resistant mice sensitive to irradiation. Collectively, we demonstrate how inflammatory polarization of CAFs affects cancer therapy and identify IL-1 signaling as an attractive target to repolarize CAFs in neoadjuvant therapy of rectal cancer.

overall_design: Total of 104 patients RNA samples (pre-therapeutic biopsies) belonging to patients of pathological complete and non complete response post radiotherapy. Addiotional 12 RNA samples from post chemo-RT surgical materials of RCA patients.

first: Emmanouil

last: Fokas

first: Adele

last: Nicolas


title: Tet2 deletion in CD4+ T cells disrupts Th1 lineage commitment in memory cells and enhances T follicular helper cell recall responses

city: Salt Lake City

first: Jeffrey

middle: Scott

last: Hale

state: Utah

address: 15 North Medical Drive

department: Pathology

institute: University of Utah

type: Methylation profiling by high throughput sequencing

summary: Following viral clearance, antigen-specific CD4+ T cells contract and form a pool of distinct Th1 and Tfh memory cells that possess unique epigenetic programs, allowing them to rapidly recall their specific effector functions upon rechallenge. DNA methylation programing mediated by the methylcytosine dioxygenase Tet2 contributes to balancing Th1 and Tfh cell differentiation during acute viral infection, however the role of Tet2 in CD4+ T cell memory formation and recall is unclear. Using adoptive transfer models of antigen-specific WT andTet2KO CD4+ T cells, we find that Tet2 is required for full commitment of CD4+ T cells to the Th1 lineage and that in the absence of Tet2, memory cells preferentially recall a Tfh like phenotype with enhanced expansion upon secondary challenge. These findings demonstrate an important role for Tet2 in enforcing lineage commitment and programing proliferation potential, and highlight the potential of targeting epigenetic programing to enhance adaptive immune responses.

overall_design: To determine the role of TET2 in the memory T cell recall response, we performed whole genome enzymatic methylation sequencing of either WT or Tet2 KO secondary effector cells 7 days post LCMV rechallenge and then sorted into secondary Tfh (CXCR5+) and Th1 (CXCR5-) effector subsets.

first: Andrew

last: Baessler

first: Bryce

last: Fuchs

first: Bryant

last: Perkins

middle: W

last: Richens

first: Camille

last: Novis

first: Malia

last: Harrison-Chau

first: Linda

last: Sircy

first: Kendaall

last: Thiede


title: Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

first: Thomas

last: Kehrer

address: 1 Gustave L. Levy Place

department: Microbiology

institute: Icahn School of Medicine at Mount Sinai

summary: We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon signaling pathway by directly interacting with the Nup98-Rae1 complex at the nuclear pore, and disrupting bi-directional nuclear-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We unambiguously show that ORF6 plays a key non-redundant role in the antagonism of IFN signaling by interfering with karyopherin-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian hamster model in vivo. In addition, we found that by contributing to the inhibition of cellular mRNA export, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 subvariants and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2.

overall_design: BulkRNA sequencing of A549-ACE2 cells infected with recombinant SARS-CoV-2 viruses of wildtype sequence or carrying  a deletion of the ORF6 coding sequence. All SARS-CoV-2 infections were performed under BSL3 containment in accordance with the biosafety protocols developed by the Icahn School of Medicine at Mount Sinai (ISMMS).

first: Anastasija

last: Cupic

first: Adolfo

last: Garcia-Sastre

first: Lisa

last: Miorin


title: Loss of Mtm1 causes cholestatic liver disease in a model of X-linked myotubular myopathy

status: Public on Aug 10 2023

city: San Francisco

first: Kristen

last: Seim

address: 201 Haskins Way

institute: GraphiteBio

summary: X-linked myotubular myopathy (XLMTM) is a fatal congenital disorder caused by mutations in the MTM1 gene. Currently, there are no approved treatments, though AAV8-mediated gene transfer therapy has shown promise in animal models and preliminarily in patients. However, four patients with XLMTM treated with gene therapy have died from progressive liver failure, and hepatobiliary disease has now been recognized more broadly in association with XLMTM. In an attempt to understand whether loss of MTM1 itself is associated with liver pathology, we have characterized a novel liver phenotype in a zebrafish model of this disease. Specifically, we have found that loss-of-function mutations in mtm1 lead to severe liver abnormalities including impaired bile flux, structural abnormalities of the bile canaliculus, and improper endosomal-mediated trafficking of canalicular transporters. Using a reporter tagged Mtm1 zebrafish line, we have established localization of Mtm1 in the liver in association with Rab11 and canalicular transport proteins, and demonstrated that hepatocyte specific re-expression of Mtm1 can rescue the cholestatic phenotype. Lastly, we completed a targeted chemical screen, and found that Dynasore, a dynamin II inhibitor, is able to partially restore bile flow and transporter localization to the canalicular membrane. In summary, we demonstrate for the first time liver abnormalities that are directly caused by MTM1 mutation in a pre-clinical model, thus establishing the critical framework for better understanding and comprehensive treatment of the human disease.

overall_design: Total mRNAs were extracted from pools of dissected livers from 6 dpf abcb11b mutants and WT siblings, 20 livers per replicate and 3 replicates per genotype, by using Arcturus PicoPure RNA isolation kit (Applied Biosystems, KIT0204). Samples were submitted to Novogene Co for library preparation and RNA-sequencing using the vendor’s standard protocol. Bioinformatic analysis was performed as described above.

first: Ashish

last: Deshwar

first: Sophie

last: Karolczak

first: James

last: Dowling

first: Chunyue

last: Yin

first: Jillian

last: Ellis


title: Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children

city: San Diego

first: Ashu

last: Sethi

address: 9420 Athena Circle

department: Health Sciences

institute: La Jolla Institute for Immunology

summary: Cow milk (CM) allergy is the most prevalent food allergy in young children in the US and Great Britain. Current diagnostic tests are either unreliable (IgE, skin prick test), or resource-intensive with risks (food challenges). Here we set out to determine if allergen-specific T cells in cow milk allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. Starting from cow milk extract, we mapped T cell responses to a set of reactive epitopes that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing allergic from non-allergic subjects. Using single-cell RNA and paired TCR sequencing we detected significant changes in the transcriptional program and clonality of cow milk antigen-specific (CM+) T cells elicited by the pool in allergic vs. non-allergic subjects ex vivo. CM+ T cells from allergic subjects had increased percentages of FOXP3+ over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells (Tregs) that have suppressive activity, but CM+ FOXP3+ cells from allergic subjects showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared to non-allergic subjects, suggesting that these are not conventional Tregs. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to utilize surface markers (CD25, CD127, CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from CMA subjects in an independent cohort, which could be used for diagnostic purposes. Looking specifically for Th2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in CMA subjects and that had high expression of Th2 cytokines and pathogenic Th2/Tfh markers. Overall, these findings suggest that there are several differences in the phenotype of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.

overall_design:
Antigen,specific,CD4,memory,T,cells,from,cow,milk,allergic,and,non-allergic,children,analyzed,by,scRNA/TCR-Seq
---------------------------------------
Authors state:
The data in this study came from Human samples, who did not approve public sharing of their sequencing data. Therefore we can not upload Raw files.

first: Sloan

middle: A

last: Lewis

first: Aaron

last: Sutherland

first: Ferran

last: Soldevilla

first: Luise

last: Westernberg

first: Minori

last: Aoki

first: April

last: Frazier

first: Synaida

last: Maiche

first: Mich

last: Erlewyn-Lajeunesse

first: Hasan

last: Arshad

first: Stephanie

last: Leonard

first: Susan

last: Laubach

first: Jennifer

last: Dantzer

first: Robert

last: Wood

first: Alessandro

last: Sette

first: Gregory

last: Seumois

first: Pandurangan

last: Vijayanand

first: Bjoern

last: Peters


title: Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment

city: Tokyo

first: Tomohiro

last: Yabushita

address: 7-3-1, Hongou, Bunkyo-ku

institute: The University of Tokyo

summary: Decitabine (DAC) is used clinically for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To elucidate its exact mechanism of action, we performed a genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells and revealed that mitotic regulation plays a pivotal role in DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations and antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, the overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to perturb mitosis through aberrant DNMT1-DNA covalent bonds. This clinically revised mode of action is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway.

overall_design: Our genome-wide CRISPRa screen revealed the enrichment of genes related to cholesterol metabolism, such as Srebf2, Ldlr, Cyp51 and Pcyt2, in decitabine resistance. Statins are one of the most widely used drugs worldwide and suppress endogenous cholesterol synthesis by inhibiting HMG-CoA reductase. We found that both simvastatin and lovastatin synergized with DAC to inhibit the growth of HL-60 cells and Kasumi-1 cells in several reference models. Therefore, We next performed RNA-seq of HL-60 treated with DAC, simvastatin or both.

first: Tomoya

last: Isobe

first: Toshio

last: Kitamura

first: Susumu

last: Goyama


title: Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes

city: La Jolla

first: Hunter

last: Bennett

address: 9500 Gilman Drive, MC 0651

department: Dept. of Cellular & Molecular Medicine

institute: University of California, San Diego

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


title: Chromatin mechanism of DELLA-mediated gene repression and Gibberellin-induced gene activation in rice

first: Qi

last: Li

address: Lion Rock Street

department: Hongshan District

institute: Huazhong Agricultural University

summary: GA treatment of wild-type rice and use of DELLA protein mutant for RNA-seq and ChIP-seq. Explaining the chromatin mechanisms of DELLA-mediated gene repression and GA signalling-induced gene activation.

overall_design: To investigate the chromatin mechanism of DELLA-mediated gene repression. We performed H3K27me3 ChIP-seq analysis and transcriptome analysis using 12-day-old mutant seedlings of DELLA and the corresponding wild-type seedlings. To study the GA-induced genome-wide histone modification changes, we performed H3K27me3 ChIP-seq analysis of rice plants treated with 100uM GA for 6h and then we analyzed the transcriptomes of rice seedlings treated with or without 100uM GA for1h.

first: Junjie

first: Dao-Xiu

last: Zhou


title: Nutrient-driven dedifferentiation of enteroendocrine cells promotes adaptive intestinal growth

city: Bunkyo-ku

first: Luis

middle: Augusto Eijy

last: Nagai

state: Tokyo

address: #201 Main building, Institute for Quantitative Biosciences, 1-1-1 Yayoi

department: Institute for Quantitative Biosciences

summary: Post-developmental organ resizing improves organismal fitness under constantly changing nutrient environments. Although stem cell abundance is a fundamental determinant of adaptive resizing, our understanding of its underlying mechanisms remains primarily limited to the regulation of stem cell division. Here, we demonstrate that nutrient fluctuation induces dedifferentiation in the Drosophila adult midgut to drive adaptive intestinal growth. From lineage tracing and single-cell RNA sequencing, we identify a subpopulation of enteroendocrine (EE) cells that convert into functional intestinal stem cells (ISCs) in response to dietary glucose and amino acids by activating the JAK-STAT pathway. Genetic ablation of EE-derived ISCs severely impairs ISC expansion and midgut growth despite the retention of resident ISCs, and in silico modeling further indicates that EE dedifferentiation enables an efficient increase in the midgut cell number while maintaining epithelial cell composition. Our findings identify a physiologically induced dedifferentiation that ensures ISC expansion during adaptive organ growth in concert with nutrient conditions.

overall_design: Cells in the adult Drosophila midgut were collected from 1-day and 3-day old female adults and analysed by scRNA-seq.

first: Hiroki

first: Sohei

last: Tasaki

first: Ryuichiro

last: Nakato

first: Masayuki

last: Miura

first: Yu-ichiro

last: Nakajima