title: Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease

first: Zhongwei

last: Li

address: 1425 San Pablo St

department: Keck medicine

institute: University of Southern California

summary: We developed novel chemically defined recipe media named NPSR-v2, that was capable of expanding mouse and human nephron progenitor cells (NPC) for long term culture in vitro in 2D (2 Dimension) format. NPSR-v2 and these expanded NPC can be used for versatile application, such as reprograming, whole genome-wide CRISPR screen, kidney-associated disease modeling and library drug screening etc.

sample_organism:
Homo sapiens
Mus musculus
synthetic construct

overall_design: Comparative gene expression profiling analysis of RNA-seq data for NPSR-v2 long-term cultured mouse and human nephron progenitor cells and primary mouse and human nephron progenitor cells. We performed bulk RNA-Seq analysis using hNPC signature genes to explore the similarity among reprogrammed iNPCLCs, cultured iNPCs (in v2) and primary hNPCs. To investigate the mechanism underlying the reprogramming of podocytes into iNPCLCs with hNPSR-v2 medium, podocytes cultured with hNPSR-v2 were collected at time-course for bulk RNA-Seq analysis. And nephron organoids derived following the advanced nephron organoids induction protocol were collected at time-course for bulk RNA-Seq analysis to investigate the iNPC-derived nephron organoids development process.

first: Biao

last: Huang

first: Zipeng

last: Zeng


title: ATRX guards against aberrant differentiation in mesenchymal progenitor cells (RNA-Seq)

status: Public on Mar 13 2024

city: New York

first: Douglas

last: Barrows

state: NY

address: 1230 York Ave

country: USA

institute: Rockefeller University

type: Expression profiling by high throughput sequencing

summary: Alterations in the tumor suppressor ATRX are recurrently observed in several cancer types including sarcomas, which are mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors (Pparγ and Cebpα) and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks at putative enhancer elements and promoters.  Finally, we observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements.  Our results demonstrate ATRX functions to buffer against differentiation in mesenchymal progenitor cells, which has implications for understanding ATRX loss of function in sarcomas.

overall_design: We determined genes that changes in expression with the expression of ATRX deficiency in mesenchymal progenitor cells, using RNA-seq followed by gene exrepssion analyses.

first: Yan

last: Fang

first: Yakshi

last: Dabas

first: Thomas

middle: S

last: Carroll

first: William

middle: D

last: Tap

first: Benjamin

middle: A

last: Nacev


title: The intestinal microbiota modulates the transcriptional landscapes of iNKT cells

status: Public on Mar 14 2024

city: Toronto

first: Thierry

middle: Robert

last: Mallevaey

state: ON

address: 1 King's College Circle

department: Immunology

country: Canada

institute: University of Toronto

summary: Invariant Natural Killer T (iNKT) cells are unconventional T cells that respond to microbe-derived glycolipid antigens. iNKT cells exert fast innate effector functions that regulate immune responses in a variety of contexts, including during infection, cancer, or inflammation. The roles these unconventional T cells play in intestinal inflammation remain poorly defined and vary based on the disease model and species. Our previous work suggested that the gut microbiota influenced iNKT cell functions during dextran sulfate sodium-induced colitis in mice. This study, shows that iNKT cell homeostasis and response following activation are altered in germ-free mice. Using prenatal fecal transplant in specific pathogen-free mice, we show that the transcriptional signatures of iNKT cells at steady state and following αGC-mediated activation in vivo are modulated by the microbiota. Our data suggest that iNKT cells sense the microbiota at homeostasis independently of their T cell receptors. Finally, iNKT cell transcriptional signatures are different in male and female mice. Collectively, our findings suggest that sex and the intestinal microbiota are important factors that regulate iNKT cell homeostasis and responses. A deeper understanding of microbiota-iNKT cell interactions and the impact of sex could improve the development of iNKT cell-based immunotherapies.

overall_design: Transcriptional analysis of iNKT cells isolated from the mesenteric lymph nodes (mLN) at 24 h after oral gavage of PBS and alpha-galactosylceramide (aGC) in C57BL/6J mice that were born to dams that received fecal transplant (FT) from wild-type (WT) or CD1d-knockout (KO) donor mice.

first: Qiaochu

last: Lin

first: Meggie

last: Kuypers

first: Yuriy

last: Baglaenko

first: Eric

last: Cao

first: Kebria

last: Hezaveh

first: Tijana

last: Despot

first: Carolina

last: de Amat Herbozo

first: Mayra

last: Cruz Tleugabulova

first: Juan

last: Mauricio Umaña

first: Tracy

middle: L

last: McGaha

first: Dana

middle: J

last: Philpott


title: ATRX guards against aberrant differentiation in mesenchymal progenitor cells [ATAC-seq]

first: Douglas

last: Barrows

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: We determined the chromatin accessibilities in genome-wide scale in undifferentiated pleomorphic sarcoma cell line with ATRX WT or loss of ATRX.

first: Yan

last: Fang

first: Sam

last: Singer


title: Engineered CD47 protects T cells for enhanced antitumor immunity

city: Stanford

first: Yiyun

last: Chen

state: California

address: 265 Campus Dr Room G2105

department: Stanford Cancer Institute

institute: Stanford University

summary: Adoptively transferred T cells and agents designed to block the CD47/SIRPalpha axis are promising cancer therapeutics that activate distinct arms of the immune system. We administered anti-CD47 with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which abrogated therapeutic benefit. anti-CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRPalpha and provided a “don’t-eat-me” signal that was not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following anti-CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy synergistically enhanced antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests enhanced potency against solid tumors.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Orthotopic 143B osteosarcoma human tumors were established in NSG mice. Mice were treated with either no T cells, mock transduced T cells, CD47 WT (47WT-), or engineered CD47 (47E-) expressing Her2.BBz human CAR T cells, each ± the anti-humanCD47 antibody B6H12 (8 experimental samples total, pooled from 3 mice per treatment group; n = 24 mice). Dissociated tumors from this 143B experiment were sorted for live cells using a Live/Dead stain at the Stanford Shared FACS facility. Single-cell RNAseq libraries were prepared using the Chromium Next GEM Single Cell 5' v2 platform (10x GENOMICS). Libraries were sent to Novogene for sequencing on a NovoSeq S4 lane (PE150) with approximately 30,000 mean reads per cell.

first: Sean

last: Yamada-Hunter

first: Katherine

last: Freitas

first: Crystal

last: Mackall


title: Effect of group III phospholipase A2 deletion on gene expression in bronchial epithelial cells in an antigen-induced asthma model.

city: Tokyo

first: Yoshitaka

last: Taketomi

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

department: CDBIM, Graduate School of Medicine,

country: Japan

institute: The University of Tokyo

summary:
Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of airway obstruction, hyperresponsiveness, remodeling, and eosinophilia. Phospholipase A2s (PLA2s), which release fatty acids and lysophospholipids from membrane phospholipids, have been implicated in exacerbating asthma by generating pro-asthmatic lipid mediators, but an understanding of the association between individual PLA2 subtypes and asthma is still incomplete. Here, we show that group III secreted PLA2 (sPLA2-III) plays an ameliorating, rather than aggravating, role in asthma pathology. In both mouse and human lungs, sPLA2-III was expressed in bronchial epithelial cells and decreased during the asthmatic response. In an ovalbumin (OVA)-induced asthma model, Pla2g3 knockout (-/-) mice exhibited enhanced airway hyperresponsiveness, eosinophilia, OVA-specific IgE production, and type 2 cytokine expression as compared to Pla2g3 wild-type (+/+) mice. Lipidomics analysis showed that the pulmonary levels of several lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (LPA), were decreased in OVA-challenged Pla2g3-/- mice relative to Pla2g3+/+ mice. LPA receptor 2 agonists suppressed thymic stromal lymphopoietin expression in bronchial epithelial cells and reversed airway hyperresponsiveness and eosinophilia in Pla2g3-/- mice, suggesting that sPLA2-III negatively regulates allergen-induced asthma at least by producing LPA. Thus, the activation of the sPLA2-III-LPA pathway may be a new therapeutic target for allergic asthma.
Microarray gene profiling of the bronchial epithelial cells isolated from OVA-challenged lung revealed the increased expression of various chemokines and type 2 epithelial cytokines in bronchial epithelial cells from Pla2g3-/- mice as compared to those from Pla2g3+/+ mice following OVA challenge.

overall_design: Model, antigen (OVA)-induced asthma. Mice were sensitized with intraperitoneal injections of ovalbumin (10 µg) plus 1.3 mg of aluminum hydroxide (alum adjuvant) on days 0 and 14. On days 26, 27, and 28, the mice were challenged with 1% OVA aeroallergen for 30 min per day.; Cells, bronchial epithelial cells isolated from mouse lung; Groups, Pla2g3 knockout (–/–) and wild-type (+/+); Condiotion, 3 h after final antigen challenge versus control; RNA, total RNA samples pooled from four samples.

first: Asako

last: Hamu-Tanoue

first: Yasumasa

last: Nishito

first: Hiromasa

last: Inoue

first: Makoto

last: Murakami


title: Microcurrent-Mediated Modulation of Myofibroblasts for Cardiac Repair and Regeneration

city: Berlin

first: Dipthi

last: Bachamanda Somesh

address: Robert-Rössle Str. 10

country: Germany

institute: Berlin heals

summary: Myofibroblasts are one of the key cell types in cardiac injuries, leading to scar tissue formation and reduced heart function. To investigate the impact of microcurrent treatment on these cells, primary rat cardiac fibroblasts were initially differentiated into myofibroblasts. Subsequently, the differentiated myofibroblasts were subjected to microcurrent treatment at 1 and 2 µA/cm2 direct current (DC) with and without polarity reversal. Microcurrent treatments resulted in distinct transcriptional signatures and improved cell behavior. The observations show signs of microcurrent-mediated reversal of myofibroblast phenotype, possibly reducing cardiac fibrosis, and providing insights for cardiac tissue repair.

overall_design: Four sample groups were analyzed: (1) rat neonatal cardiac fibroblasts (CF, starting cells), (2) rat myofibroblasts (MF, differentiated from cardiac fibroblasts), (3) differentiated myofibroblasts treated with 1 μA/cm2 microcurrents (1μA) and (4) differentiated myofibroblasts treated with 2 μA/cm2 microcurrents (2μA). Two biological replicates were included for each sample group.

first: Karsten

last: Jürchott

first: Yadhu

last: Kumar

first: Peter

last: Göttel

first: Johannes

last: Müller


title: Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins [scRNAseq]

city: Philadelphia

first: Jesse

last: Lee

state: PA

address: South Pavilion, Room 8-326

institute: University of Pennsylvania

summary: We considered the possibility that STAT3 signaling specifically in hepatocytes may influence T cell surveillance in cancer

overall_design: Transcriptomic profiling on tumors from tumor bearing WT and Alb-Cre+/+;Stat3f/f mice.

first: Meredith

last: Stone


title: Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins [Tumor]


title: ATRX guards against aberrant differentiation in mesenchymal progenitor cells (CUT&RUN)

first: Douglas

last: Barrows

overall_design: Characterization of the genome-wide incoporation of histone posttranslational modifications and ATRX in mesenchymal progenitor cells.

first: Yan

last: Fang

first: Yakshi

last: Dabas

first: Thomas

last: Carroll

first: William

last: Tap

first: Benjamin

last: Nacev


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [NTD-Seq]

status: Public on Mar 12 2024

city: NEW HAVEN

first: MANOJ

last: PILLAI

state: CT

address: 300 GEORGE STREET #786

institute: YALE UNIVERSITY

summary:
To investigate changes in RNAPII distribution in response to SF3B1 mutations, we peformed ChIP-seq using antibody against N-terminal domain of RNAPII (Pan-Pol2) in K562 cells
We then performed coverage plot analysis using data obtained from ChIP-seq to investigate RNAPII distribution changes

overall_design: ChIP-seq against NTD-RNAPII in K562 cells across different conditions

first: Boddu

last: Prajwal

first: Pillai

last: Manoj


title: ATRX guards against aberrant differentiation in mesenchymal progenitor cells

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

overall_design: Refer to individual Series


title: Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins [Liver]

summary: We sought to understand the mechanism by which hepatocytes coordinate immune surveillance

overall_design: Transcriptomic profiling on livers from control WT mice, tumor bearing WT mice, and tumor bearing Alb-Cre+/+;Stat3f/f mice.


title: Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Murine ChIP-Seq]

summary:
To investigate changes in Pol2 distribution in response to SF3B1 mutations, we peformed ChIP-seq using antibody against Pol2 (both CTD and elongation specific (Ser2P and Ser5p). For the murine model, SF3B1 mutant (and WT counterparts) were treated with tamoxifen daily x 5 days at 5 weeks of age. Mice were euthanized at end of week 7 and bone marrow was harvested to isolate lineage negative cells
We then performed coverage plot analysis using data obtained from ChIP-seq from IP fractions to investigate Pol2 distribution changes

overall_design: ChIP-seq against Pol2 in murine lineage negative marrow cells (wild type vs SF3B1K700E mutant) at 2 weeks post induction


title: Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins [HotvsCold]

summary: To define changes in the liver that coordinate systemic inflammation and T cell surveillance, we performed RNA sequencing on livers of mice with T cellhigh and T celllow tumors.

overall_design: Transcriptomic profiling on livers from T celllow and T cellhigh tumor-bearing mice


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Ser5PRNAPII ChIP-Seq]

summary:
To investigate changes in the elongating form of RNA Polymerase II across different conditions, we peformed ChIP-seq using antibody against the Ser5P RNAPII of pTEFb in K562 cells at 4 days after modified allele expression
We then performed coverage plot analyses using data obtained from ChIP-seq from IP and Input fractions to investigate Ser5P RNAPII distribution changes

overall_design: ChIP-seq against Ser2P RNAPII in K562 cells across different conditions at 4 days post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [CHD ChIP-Seq]

summary:
To investigate changes in the CHD1 distribution across different conditions, we peformed ChIP-seq using antibody against the CHD1 in K562 cells at 4 days after modified allele expression
We then performed coverage plot analyses using data obtained from ChIP-seq from IP fractions to investigate CHD1 distribution changes

overall_design: ChIP-seq against CHD1 in K562 cells across different conditions at 4 days post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [ATAC-Seq]

summary:
To investigate changes in chromatin landscape and nucleosome positioning in response to SF3B1 mutations, we peformed ATAC seq in K562, human CD34, and murine cKit+,Sca1+,Lin- cells
We then performed peak-based and nucleosome positioning analyses using data obtained from ATAC to investigate changes in chromatin accessibility and promoter nucleosome occupancy, respectively

overall_design: ATAC-seq in WT and SF3B1K700E K562 cells at 4 days post doxycycline induction, along with human CD34+ (isolated from healthy volunteer and MDS patient samples) and murine cKit+,Sca1+,Lin- cells (from SF3B1WT vs SF3B1K700E expressing mice)


title: Disruption of RNAPII transcription elongation links Oncogenic SFB31 mutations to replciation stress and targetable alterations in chromatin landscape [mNET-Seq]

summary:
To investigate changes in RNAPII distribution in response to SF3B1 mutations, we peformed mNET-seq using antibody against either pan Pol II CTD or Ser2P Pol II CTD in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed coverage plot analysis using data obtained from mNET-seq to investigate RNAPII distribution changes. Drosophila spike-in was included for the pan-Pol II CTD studies.

overall_design: mNET-seq against RNAPII in K562 (wild type vs SF3B1K700E mutant) cells at 4 days, post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [H3K36me3 ChIP-Seq]

summary:
To investigate changes in the H3K36me3 distribution across different conditions, we peformed ChIP-seq using antibody against the H3K36me3 in K562 cells at 4 days after modified allele expression
We then performed coverage plot analyses using data obtained from ChIP-seq from IP  fractions to investigate H3K36me3 distribution changes

overall_design: ChIP-seq against H3K36me3 in K562 cells across different conditions at 4 days post doxycycline induction


title: Engrailed 1 deficiency induces changes in ciliogenesis during human neuronal differentiation

city: Neuherberg

first: Sebastian

last: Schmidt

address: Ingolstädter Landstraße 1

department: Institute of Developmental Genetics

institute: Helmholtz Zentrum München

summary: A key pathological feature of Parkinson's Disease (PD) is the progressive degeneration of dopaminergic neurons (DAns) in the substantia nigra pars compacta. Considering the major role of EN1 in the development and maintenance of these DAns and the implications from En1 mouse models, it is highly interesting to study the molecular and protective effect of EN1 also in a human cellular model. Therefore, we generated EN1 knock-out (ko) human induced pluripotent stem cell (hiPSCs) lines and analyzed these during neuronal differentiation. Although the EN1 ko didn't interfere with neuronal differentiation and generation of tyrosine hydroxylase positive (TH+) neurons per se, the neurons exhibited shorter neurites. Furthermore, mitochondrial respiration, as well as mitochondrial complex I abundance was significantly reduced in fully differentiated neurons. To understand the implications of an EN1 ko during differentiation, we performed a transcriptome analysis of human neuronal precursor cells (hNPCs) which unveiled alterations in cilia-associated pathways. Further analysis of ciliary morphology revealed an elongation of primary cilia in EN1-deficient hNPCs. Besides, also Wnt signaling pathways were severely affected. Upon stimulating hNPCs with Wnt which drastically increased EN1 expression in WT lines, the phenotypes concerning mitochondrial function and cilia were exacerbated in EN1 ko hNPCs. They failed to enhance the expression of the complex I subunits NDUFS1 and 3, and now displayed a reduced mitochondrial respiration. Furthermore, Wnt stimulation decreased ciliogenesis in EN1 ko hNPCs but increased ciliary length even further. This further highlights the relevance of primary cilia next to mitochondria for the functionality and correct maintenance of human DAns and provides new possibilities to establish neuroprotective therapies for PD.

overall_design:
To investigate the effect of EN1 during dopaminergic neuron differentiation on a human background, we generated EN1 knock-out human iPSCs using CRISPR/Cas9.
In total, 3 EN1 knock-out lines and 5 wildtype lines were differentiated towards dopaminergic neurons.
Gene expression profiling was performed at the stage of neuronal precursor cells. 3 replicates per cell line were analyzed.
Neuronal precursor cells were stimulated with CHIR99021 to assess the effect of Wnt signaling and it's interaction with EN1 during dopaminergic neuron differentiation.
Comparative analysis of RNA-seq data for wildtype and EN1 knock-out lines in unstimulated and Wnt stimulated conditions.

first: Sina

last: Hembach

first: Tanja

last: Orschmann

first: Florian

last: Giesert

first: Daniela

middle: M

last: Vogt Weisenhorn

first: Wolfgang

last: Wurst


title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Primary and secondary cone photoreceptor cell death in retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, leads to severe vision impairment and blindness. Regardless of the fact that protection of cone photoreceptor cells under stress conditions, such as retinal degenerative diseases, is crucial for maintaining vision, the underlying molecular mechanisms are unclear. Here, we investigated the function of the deubiquitinase Otud7b/Cezanne in the retina. We identified that Otud7b is predominantly expressed in photoreceptor cells in the mouse retina. While the ablation of Otud7b did not cause a significant defect in development and maturation of the mouse retina, Otud7b‒/‒ mice subjected to light-induced damage, which is one of the dry age-related macular degeneration models, exhibited increased cone photoreceptor degeneration. In addition, Otud7b deficiency in Mak‒/‒ mice, a retinitis pigmentosa mouse model, resulted in further cone photoreceptor degeneration. Moreover, neuronal cells deficient in Otud7b were susceptible to serum starvation, resulting in cell death. We found that NF-κB activity is increased in the Otud7b‒/‒ retinas exposed to light by RNA-sequencing analysis. Luciferase reporter assay also demonstrated increased NF-κB activation in Otud7b-deficient neuronal cells under stress. The neuronal cell death resulting from Otud7b deficiency was suppressed through the inhibition of NF-κB. Furthermore, we observed that inhibition of NF-κB attenuated cone photoreceptor degeneration in the light-exposed Otud7b‒/‒ retina. Together, the current study suggests that Otud7b deubiquitinase protects cone photoreceptor cells under stress conditions by modulating the NF-κB activity.

overall_design: We performed RNA-seq analysis using retinal RNAs from Otud7b+/+ and Otud7b‒/‒ mice that underwent light exposure.

first: Leah

middle: R

last: Varner

first: Taro

last: Chaya

last: Okuzaki

first: Takahisa

last: Furukawa


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [INTS-11 ChIP-Seq]

summary:
To investigate changes in the INTS-11 distribution across different conditions, we peformed ChIP-seq using antibody against INTS11 in K562 cells at 4 days after modified allele expression
We then performed coverage plot analyses using data obtained from ChIP-seq from IP  fractions to investigate INTS11 distribution changes

overall_design: ChIP-seq against INTS11 in K562 cells across different conditions at 4 days post doxycycline induction


title: A phage nucleus-associated RNA-binding protein required for jumbo phage infection

city: La Jolla

first: Brian

last: Yee

state: CA

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: Large-genome bacteriophages (jumbo phages) of the Chimalliviriadae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA. Targeted knockdown of ChmC using mRNA-targeting Cas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

overall_design: eCLIP-seq (enhanced UV crosslinking and immunoprecipitation, followed by deep sequencing) in PhiPA3-infected P. aeruginosa cells expressing GFP-tagged ChmC.

first: Eray

last: Enustun

first: Emily

last: Armbruster

first: Jina

last: Lee

first: Sitao

last: Zhang

first: Yajie

last: Gu

first: Amar

last: Deep

first: Jack

last: Naritomi

first: Qishan

last: Liang

first: Stefan

last: Aigner

first: Vorrapon

last: Chaikeeratisak

first: Don

last: Cleveland

first: Majid

last: Ghassemian

first: Gene

middle: W

last: Yeo

first: Joe

last: Pogliano

first: Kevin

last: Corbett


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [DRIP-seq]

first: MANOJ

last: PILLAI

summary:
To investigate the distribution of R-loop formation, we peformed R-loop mapping analysis using S9.6 antibody-based DRIP-seq in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed metaplot and peak-based analysis using data obtained from DRIP-seq

overall_design: R-loop analysis for K562 (wild type vs SF3B1K700E mutant) cells

first: Boddu

last: Prajwal

first: Pillai

last: Manoj


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [GRO-seq]

summary:
To investigate redistribution of RNAPII in response to the SF3B1 mutation and to study its effects on nascent gene expression, we peformed fastGRO-seq (variant of GRO-seq using 4-thio-UTP based nuclear run-on) in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed metaplot-based and gene expression analysis using data obtained from fastGRO-seq

overall_design: Nuclear run-on assay for K562 (wild type vs SF3B1K700E mutant) cells at 4 days, post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [TT-TL seq]

summary:
To investigate how whether the RNAPII defect is premature pause release defect or gene body elongation defect  due to splicing factor mutations, we peformed TT-TL-seq (TT seq with a time lapse component) in K562 cells (wild type and SF3B1K700E mutant; U2AF1wild type vs U2AF1 S34F mutant) at 4 days after mutant allele expression
We then performed metaplot-based and gene expression analysis using data obtained from TT-TL seq

overall_design: 5-min 4sU pulse in K562 (SF3B1 wild type vs SF3B1K700E mutant) as well as (U2AF1wild type vs U2AF1 S34F mutant)  cells at 4 days, post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [RNA-seq]

summary:
To investigate the impact of altered transcription and chromatin on steady state transcript levels, we peformed gene expression and splicing analysis in K562 cells (wild type and SF3B1K700E mutant) at 4 and 8 days after mutant allele expression
We then performed gene expression profiling analysis using data obtained from RNA-seq  at two time points.

overall_design: Differential gene expression and splicing analysis for K562 (wild type vs SF3B1K700E mutant) cells


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [nascent RNAseq]

summary:
As a complementary approach to GROseq, we peformed short read sequencing of nascent RNA (nSRS-seq) in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed nascent gene transcription expression using data obtained from nSRS-seq

overall_design: Nascent/Chromatin bound RNA differential gene expression in K562 (wild type vs SF3B1K700E mutant) cells


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [CDK9-chIP-seq]

summary:
To investigate changes in p-TEFb distribution in response to SF3B1 mutations, we peformed ChIP-seq using antibody against the CDK9 component of pTEFb in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed coverage plot analysis using data obtained from ChIP-seq from IP and Input fractions to investigate CDK9 distribution changes

overall_design: ChIP-seq against CDK9 in K562 (wild type vs SF3B1K700E mutant) cells at 4 days, post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [NTD-chIP-seq]

summary:
To investigate changes in RNAPII distribution in response to SF3B1 mutations, we peformed ChIP-seq using antibody against N-terminal domain of RNAPII (Pan-Pol2) in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed coverage plot analysis using data obtained from ChIP-seq to investigate RNAPII distribution changes

overall_design: ChIP-seq against NTD-RNAPII in K562 (wild type vs SF3B1K700E mutant) cells at 4 days, post doxycycline induction


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [ATAC-seq]


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [CUT&RUN]

summary:
To investigate changes in the histone marks and RNA Polymerase II across different conditions, we peformed CUT&RUN seq using antibody against H3K4me3, H3K27me3, H3K27ac, and H3K4me1 in K562 cells at 4 days after modified allele expression, or H3K4me3 and Ser2P RNAPII in human CD34 cells
We then performed coverage plot analyses using data obtained from CUT&RUN to investigate changes in histone marks or Ser2P RNAPII

overall_design: CUT&RUN-seq against H3K4me3, H3K27ac, H3K27me3, H3K4me1 in WT and SF3B1K700E K562 cells at 4 days post doxycycline induction and CUT&RUN-seq against H3K4me3 and Ser2P RNAPII in CD34 cells obtained either from SF3B1 MDS patient samples or aged-matched healthy volunteers


title: Gene regulatory basis of bystander activation in CD8+ T cells (human scRNA-seq)

status: Public on Mar 11 2024

city: Ithaca

first: Andrew

last: Grimson

address: 445 Biotech Bldg

department: Molecular Biology and Genetics

institute: Cornell University

summary: CD8+ T cells have been categorized as an adaptive lymphocyte, based on their ability to recognize specific foreign antigens and mount memory responses.  However, neonatal and adult CD8+ T cells are derived from distinct HSC progenitors, which may alter the roles they play during infection.  Here, we demonstrate that neonatal CD8+ T cells can be activated by innate cytokines alone and protect the host from a variety of unrelated pathogens in the absence of TCR signaling. Using a multi-omics approach, we found that neonatal CD8+ T cells are highly responsive to innate cytokines because they can rapidly undergo chromatin remodeling, resulting in the usage of a distinct set of enhancers that are more commonly found in innate-like T cells.  We also identified a key gene regulatory axis (Bach2/AP1) that is differentially utilized by neonatal and adult CD8+ T cells to toggle their responsiveness to innate cytokines.  Importantly, the developmental switch between innate and adaptive functions in the CD8+ T cell compartment is not mediated by a change in the maturation of individual cells along a linear axis, but rather by changes in the abundance of distinct subsets of cells. Collectively, our findings provide support for the layered immune hypothesis and indicate that the CD8+ T cell compartment is more phenotypically diverse than previously thought.

overall_design: CD8+ cells were magnetically enriched by positive selection (anti-human CD8 microbeads; Miltenyi Biotech) from rested PBMCs or CBMCs. Isolated cells were then treated with IL-2 with or without co-treatment with IL-12 and IL-18 and analyzed using scRNAseq.

first: Neva

middle: B

last: Watson

first: Ravi

middle: K

last: Patel

first: Connor

last: Kean

first: Brian

last: Rudd


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Ser2PRNAPII ChIP-seq]

summary:
To investigate changes in the elongating form of RNA Polymerase II across different conditions, we peformed ChIP-seq using antibody against the Ser2P RNAPII of pTEFb in K562 cells at 4 days after modified allele expression
We then performed coverage plot analyses using data obtained from ChIP-seq from IP and Input fractions to investigate Ser2P RNAPII distribution changes

overall_design:
ChIP-seq against Ser2P RNAPII in K562 cells across different conditions at 4 days post doxycycline induction

The IP files and their corresponding input files were used to generate a common *.bw (bigwig) file.
Please note that processed data files assocaited with multiple samples are linked to the Series records and indicated in the corresponding sample description field.


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Long read sequencing]

summary:
To understand how SF3B1K700E impacts co-transcriptional splicing, we peformed long read sequencing of RNA (nSRS-seq) in K562 cells (wild type and SF3B1K700E mutant) at 4 days after mutant allele expression
We then performed co-transcriptional splicing efficiency analysis using data obtained from Long read sequencing

overall_design: Co-transcriptional splicing analysis using Nascent/Chromatin bound RNA by Pacbio Long read sequencing in K562 (wild type vs SF3B1K700E mutant) cells


title: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (Tn5 mouse)

city: STANFORD

first: Katja

last: Pekrun

address: 269 Campus Drive

department: Pediatrics & Genetics

institute: Stanford School of Medicine

summary: We investigated long-term human coagulation Factor IX expression of a novel variant when delivered into mice and rhesus macaques and compare transduction efficiencies using two different AAV capsids. In hemophilic mice injected with KP1-packaged rAAV expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared to wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months post-administration, while one animal from each group lost huFIX mRNA and protein expression over time despite comparable vector copies. We investigated if epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques to those in mice injected with the same vector, the activating histone marks were starkly reduced in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

overall_design: Mice were injected with rAAV packaged with the KP1 capsid, nuclei were isolated, and Cut&Tag analysis was performed

first: Calvin

last: Stephens

first: Adriana

last: Gonzalez-Sandoval

first: Aranyak

last: Goswami

first: Feijie

last: Zhang

first: Alice

middle: F

last: Tarantal

first: Grant

last: Blouse

first: Mark

last: Kay


title: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (Tn5 NHP)

overall_design: Rhesus macaques were injected with rAAV packaged using different capsids, nuclei were isolated, and Cut&Tag analysis was performed


title: Spatiotemporal lineage derepression by cell crowding-induced ETV4 inactivation

city: Pohang-si

first: Seungbok

last: Yang

address: 47, Jigok-ro

country: South Korea

institute: POSTECH

summary: All extant amniotes employ a single-layered epithelium of epiblasts as a starting material for gastrulation, suggesting the necessity of the epithelial structure for three germ layer derivation. Using a human embryonic stem cell (hESC) epithelium as a model system, we found that local epithelial crowding derepresses the neuroectoderm fate by spatiotemporal inactivation of ETV4. ETV4 serves as a genetic toggle switch that links cell density to lineage fates. Mechanistically, cell crowding blocks FGF receptor endocytosis by reduced cell-extracellular matrix (ECM) interaction. Disrupted endocytosis decreases ETV4 protein stability by ERK inactivation. Mathematical modeling of epithelial crowding demonstrates that the cooperativity of integrin-ECM interaction transforms the gradient of crowdedness into bistable ETV4 transition, which ensures the switch-like function of ETV4 in lineage determination. Our results propose local cell crowding in a stem cell epithelium as a key cellular mechanism for spatiotemporal regulation of lineage fates.

overall_design: mRNA profiles of ETV4 +/- and low/high density H9 hESCs.

first: Jiwon

last: Jang


title: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques

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

sample_organism:
Macaca mulatta
Mus musculus

platform_organism:
Macaca mulatta
Mus musculus

overall_design: Refer to individual Series


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF (ChIP-Seq)

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediate these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes cause only moderate effects on the chromatin landscape, while removal of the NURF specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility bound CTCF is nevertheless impaired in function resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function.

overall_design: ChIP-seq in mouse Embryonic Stem Cells, in wild-type and Bptf knock-out (2 biological replicates). IP BPTF with respective input samples.

first: Mario

last: Iurlaro

first: Francesca

last: Masoni

first: Lukas

last: Burger

last: Schübeler


title: Combining TIGIT blockade with MDSC inhibition hinders breast cancer bone metastasis by activating anti-tumor immunity

city: Tel Aviv

first: Neta

last: Erez

address: TAU

department: Pathology

country: Israel

institute: Tel Aviv University

summary: Bone is the most common site of breast cancer metastasis. Bone metastasis are incurable and are associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that crosstalk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the pro-inflammatory cytokine IL-1b as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating anti-tumor immunity. Analysis of patient samples revealed that TIGIT and IL-1b are prominent in human bone metastasis. Our findings suggest that co-targeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis.

overall_design: Granulocytes, T helper, T cytotoxic and T regulatory cells were isolated by cell sorting from normal BM, early-stage metastatic BM and from late metastatic stage of mice as described above. At late metastatic stage, two distinct sites were analyzed: the peripheral BM and the bone metastatic core of the same mouse. MARS-seq protocol was used to generate libraries. MARS-seq libraries were sequenced using Illumina NovaSeq 6000.

first: Lea

last: Monteran

first: Nour

last: Ershaid


title: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (CnT mouse)

first: Katja

last: Pekrun

last: Stephens


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF (RNA-Seq)

overall_design: RNAseq in mouse Embryonic Stem Cells, in wild-type and Snf2l knock-out (duplicates). All samples processed in the same batch.


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF (ATAC-Seq)

overall_design: ATACseq in mouse Embryonic Stem Cells, in wild-type and Snf2l (Smarca1) knock-out (duplicates). All samples processed in the same batch.


title: Following the p63/Keratin5 Basal Cells in the Sensory and Nonsensory Epithelia of The Vomeronasal Organ

city: Albany

first: Paolo

middle: Emanuele

last: Forni

address: 1400 Washington Avenue

department: Biology

institute: University at Albany

summary: The Vomeronasal organ (VNO) is a part of the accessory olfactory system, which is responsible for detecting pheromones, chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons (OSNs) in cellular structure, receptor expression, and connectivity.  The vomeronasal organ of rodents comprises a sensory epithelium and a thin nonsensory epithelium that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE.  In addition to these, the MOE also comprises p63 positive horizontal basal cells (HBCs), a second pool of quiescent stem cells that become active in response to injury.  Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14 and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of the VNO forming from progenitors along the basal lamina oft the marginal zones. Moreover, these experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

overall_design: Whole postnatal vomeronasal organs were dissected from P21 wild-type mice, cells were isolated then analyzed using scRNAseq.

first: Noah

last: LeFever

first: Raghu

last: Katreddi

first: Nikki

last: Dolphin

first: Nick

last: Mathias

middle: E


title: Yin Yang 1 regulates cohesin complex protein SMC3 in mouse hematopoietic stem cells

city: Madison

first: Xuan

last: Pan

state: WI

address: 4470 Veterinary Medicine Building

institute: University of Wisconsin

summary: Yin Yang 1 (YY1) and Structural Maintenance of Chromosomes 3 (SMC3) are two critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). While YY1 plays critical functions in promoting hematopoietic stem cell (HSC) self-renewal and maintaining HSC quiescence, SMC3 is required for proper myeloid lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and impact hematopoiesis. We found that YY1 physically interacts with SMC3 and co-occupies with SMC3 at a large cohort of promoters genome-wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species levels in HSCs, and this regulation is independent of YY1- SMC3 axis. Our results establish a distinct YY1-SMC3 axis and its impact on HSC quiescence and metabolism.

overall_design: Cleavage Under Targets and Tagmentation (CUT&Tag) targetting Yin Yang 1 (YY1) and Structural Maintenance Of Chromosomes 3 (SMC3). IgG and Trimethylation of Histone H3 at Lysine 4 (H3K4me3) were used as negative and positive controls, respectively.


title: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (CnT NHP)

first: Calvin

first: Adriana

last: Gonzalez-Sandoval

first: Aranyak

last: Goswami

first: Feijie

last: Zhang

first: Alice

last: Tarantal

first: Grant

last: Blouse

first: Mark

last: Kay


title: Long read subcellular fractionation and sequencing reveals the translational fate of full length mRNA isoforms during neuronal differentiation

city: San Francisco

first: Julie

last: Zikherman

address: 513 Parnassus Ave

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, San Francisco

summary: Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESC and NPC respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long-read sequencing-based method to study translational control.

overall_design:
Long read Frac-seq libraries in duplicate and short read Frac-seq libraries in triplicate were generated from human embryonic stem cells (ESC) and ESC-derived neural progenitor cells which were differentiated using dual-SMAD inhibition.
Long and short read Frac-seq libraries were integrated to characterize the translated transcriptome during neuronal differentiation.

first: Alexander

last: Ritter

first: Jolene

last: Draper

first: Jeremy

last: Sanford


title: Transcriptome Profiling and Characterization of Peritoneal Metastasis Ovarian Cancer Xenografts in Humanized Mice

city: Seoul

first: Sung Wan

last: Kang

address: 88, Olympic-ro 43-gil, Songpa-gu

institute: Asan Medical Center

summary: Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC.

overall_design: Humanized NSG mice were imaged using the IVIS system two weeks post-inoculation of SKOV-3 Luc cells to confirm tumor development. They were then randomly divided into two groups: a PBS control group, and a group treated with pembrolizumab. PBS and 10 mg/kg pembrolizumab were administered intraperitoneally, twice a week for three weeks. Tumor growth was monitored using the IVIS imaging system. A week after the final treatment, the mice were euthanized, and their tumor tissues were collected for RNA-seq analysis.

first: S

first: J

middle: Y

last: Lee

first: O


title: A neuro-mesenchymal cell subpopulation characterises the subaortic hematopoietic stem cell niche

city: Paris

first: Charles

last: Durand

address: 9, quai saint-Bernard

department: Laboratory of Developmental Biology

country: France

institute: Sorbonne University

summary: The first hematopoietic stem and progenitor cells (HSPCs) emerge in the embryonic dorsal aorta of midgestation mouse embryo, but the precise nature of the supportive microenvironment remains largely unexplored. To address this question, we designed a laser microdissection-based approach to isolate the dorsal and ventral tissues of the aorta at three distinct developmental stages harboring HSPCs. Bulk and single cell transcriptomics combined with computational analyses disclosed a large mesenchymal cell population in the E11.5 subaortic tissue including a subset expressing interconnected genes implicated in adhesive and neuronal functions. In vivo lineage tracing showed that this subset does not derive from neural crest cells. Histological and functional studies validated our strategy and revealed that Decorin, and Tenascin C, two components of the extracellular matrix (ECM), are essential for HSPC development. Together, our study identifies a yet unrecognized subaortic mesenchymal cell population and underscores the critical role of the ECM on embryonic HSPCs.

overall_design: E11.5 AGMs were collected, dissociated by enzymatic treatment and analyzed by the Chromium 10X Genomics v3.1 technology.

first: Pierre

last: Charbord

first: Thierry

last: Jaffredo


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [SMF]

first: Dirk

last: Schuebeler

type: Methylation profiling by high throughput sequencing

summary: Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediate these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes cause only moderate effects on the chromatin landscape, while removal of the NURF specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility bound CTCF is nevertheless impaired in function resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function.  Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function.

overall_design: Amplicon-based Single Molecule Footprinting (SMF) in mouse Embryonic Stem Cells, in wild-type and Bptf knock-out (triplicates).

first: Mario

last: Iurlaro

first: Francesca

last: Masoni

first: Lukas

last: Burger

last: Schübeler


title: Expresion data from yeast  (wild type strain and pat1 mutant) exposed to Congo Red (CR)

city: Madrid

first: Raul

last: Garcia

state: Madrid

address: Plaza Ramon y Cajal s/n

department: Microbiologia y Parasitologia

country: Spain

institute: Universidad Complutense de Madrid

summary: We did transcription profiling on the effect of PAT1 (moonlight protein whose most studied function is to degrade mRNA.) deletion in genes involved in Congo Red response (2 hours of treatment).

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: Three biological samples for wild type strain and pat1 mutant were analyzed.  Two microarray experiments were carried out for each biological sample:  1. absence of the drug   2. presence of 30 microg/ml of Congo Red for two hours


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [Hi-C]

overall_design: Hi-C in mouse Embryonic Stem Cells, in wild-type and Bptf knock-out (duplicates).

first: Ilia

last: Fliamer

first: Luca

last: Giorgetti


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [ChIP-seq]

overall_design: ChIP-seq in mouse Embryonic Stem Cells, in wild-type and Bptf knock-out (2-4 biological replicates). IP for CTCF, Rad21 and WAPL with respective input samples.


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [RNA-seq]

overall_design: RNAseq in mouse Embryonic Stem Cells, in wild-type and Rsf1, Wstf, Cecr2, Tip5, Acf1 and Bptf knock-out (duplicates or triplicates). Wild-type processed in five different batches.


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [Cut&Run]

overall_design: CUTandRUN in mouse Embryonic Stem Cells, in wild-type, and Snf2h and Bptf knock-out (duplicates). Antibodies used are against Snf2h and IgG (control).


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [Mnase-seq]

overall_design: MNase-seq in mouse Embryonic Stem Cells, in wild-type and Rsf1, Wstf, Cecr2, Tip5, Acf1 and Bptf knock-out (duplicates or triplicates). Wild-type processed in three different batches.


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF  [ATAC-seq]

overall_design: ATACseq in mouse Embryonic Stem Cells, in wild-type and Rsf1, Wstf, Cecr2, Tip5, Acf1 and Bptf knock-out (duplicates or triplicates). Wild-type processed in four different batches.


title: Single cell data from mechanically-loaded muscle

first: Daisuke

last: Motooka

summary: Skeletal muscle has abilities to regenerate after injuries and adapt to mechanical loadings. Compared with the regenerative processes, the cellular networks in the loaded muscles have not been well-explored. In this analyses, we did single-cell RNA sequencing to unveil the gene expression signature of individual cells and the cellular networks in surgically overloaded muscle.

overall_design: One side of plantaris muscles of 5 male C57BL/6J mice (12 week-old) were subjected to tenotomy and the contralateral plantaris muscles were used as the sham control. Four days after tenotomy, the plantaris muscles were pooled in each group (sham or tenotomy) digested using 0.2% collagenase type II  for 90 min. After the dilution, filtration, and centrifugation of the digested solution , the mononuclear cells were stained with Calcein AM for 5 min. Sorted Calcein AM+PI- living cells were used for scRNA-seq analyses.

first: Hayato

last: Saito

last: Okuzaki

first: So-ichiro

last: Fukada


title: Analysis of ΔcrcZ suppressor mutations

first: Rafael

last: Torres

address: Darwin 3

department: Bioinformatics for Genomics and Proteomics (BioinfoGP)

institute: CNB

summary:
The ΔcrcZ mutant shows constitutive repression of all crc/Hfq target genes in the translation. Therefore, it presents a hyper‐repressed phenotype (more repressed than the parental strain) in the acquisition of secondary carbon sources, even though the preferred carbon source is not present.
Δcrc is more sensitive to antibiotics, more susceptible to oxidative stress, and less motile than the parental strain.
The ΔcrcZ strain is more motile and more resistant to antibiotics than the parental strain. However, it presents a susceptibility profile to oxidative stress that is very similar to that of the parental strain.
When the ΔcrcZ mutant is seeded on minimal medium with a secondary carbon source it grows poorly. However, the appearance of colonies that grow similarly to the parental strain is observed. These colonies are formed from mutants derived_expression.tsv from the ΔcrcZ strain that are "revertants" of this repression phenotype in the assimilation of the secondary carbon source, and generally "pseudorevertants" of the ΔcrcZ mutation, or present a "mutation "suppressor" of the crcZ deletion. 4 mutant clones resistant to FAA (FAA resistant, present catabolic repression) and sensitive clones (FAA sensible, do not present catabolic repression) were analyzed.

overall_design:
We  performed gene expression profiling analysis using data obtained from RNA-seq of 11 different Pseudomonas aerginosa strains
Comparative gene expression profiling analysis of RNA-seq data for Pseudomonas aeruginosa PAO1, its Δcrc ΔcrcZ mutants and pseudorevertent strains from ΔcrcZ

first: F

last: Corona

last: Martinez


title: Molecular analysis of the subaortic hematopoietic stem cell niche [E12.5]

last: Durand

summary:
Hematopoietic stem cells (HSCs) are formed in the Aorta-Gonad-Mesobephros (AGM) region of mouse midgestation embryos. This process is tightly regulated in time and space by surrounding cells inculding mesenchymal cells located underneath the dorsal aorta.
We combined laser microdissection and microarrays to isolate the dorsal and ventral aortic tissues at three developmental stages and to explore their gene expression profiling.

overall_design: The dorsal and ventral aortic tissues (located in between the notochord and the roof of the dorsal aorta and underneath the dorsal aorta, respectively) were isolated by laser microdissection in the mouse embryo at three stages of AGM hematopoiesis (E10.5, E11.5 and E12.5). Microdissected tissues were collected on appropriate caps and then proceeded for RNA extraction using Acturus Picopure RNA extraction kit and Affymetrix microarrays mouse Gene 2.0 ST.

last: Charbord


title: Molecular analysis of the subaortic hematopoietic stem cell niche [E11.5]

last: Jaffredo


title: Molecular analysis of the subaortic hematopoietic stem cell niche

overall_design: Refer to individual Series


title: Molecular analysis of the subaortic hematopoietic stem cell niche [E10.5_1]


title: T-bet deficiency and Hic1 induction override TGF-b-dependency in the formation of CD103+ intestine-resident memory CD8+ T cells

status: Public on Mar 10 2024

city: Changsha

first: Liwen

last: Wang

state: Hunan

address: 138 Tongzipo road

department: Department of Hematology

country: China

institute: Third Xiangya Hospital, Central South University

summary: TGF-beta signaling is required for the differentiation of gut-resident memory CD8 T cells. Here, we showed that the deficiency of transcription factor T-bet partially rescued the differentiation of TGF-beta receptor deficient gut-resident memory CD8 T cells. Hic1 induction further strengthens TRM maturation in the absence of TGF-b and T-bet.

overall_design: Congenically marked naïve P14 TCR transgenic CD8 T cells were adoptively transferred into sex-matched C57BL/6 recipients followed by LCMV Armstrong infection. P14 T cells with different phenotypes were FACS sorted from both spleen and small intestine intraepithelial lymphocyte compartment.

first: Shruti

last: Mishra

first: Chaoyu

last: Ma

first: Nu

last: Zhang


title: Molecular analysis of the subaortic hematopoietic stem cell niche [E10.5_2]

first: Charles

first: Pierre

first: Thierry


title: Effect of deleting ultra-conserved elements PAX6_Tarzan, PBX3_Claudia on gene expression in K562 cells

city: Leiden

first: Minkang

last: Tan

address: Einthovenweg 20

department: Department of Cell and Chemical Biology

country: Netherlands

institute: Leiden University Medical Center

summary: Non-coding regulatory elements (NCRE) represent a major fraction of the human genome, play important roles in different biological pathways, and have the potential as genomic medicine targets. We developed a straightforward dual-CRISPR screening system capable of deleting thousands of NCREs genome-wide to study their functions in distinct biological contexts in K562 cells. Here we reported 4 top hits from the genome-wide screen of ultra-conserved elements (UCE), i.e. PAX6_Tarzan(chr11:31810437-31810870, hg19) and PBX3_Claudia(chr9:128457564-128457785, hg19) play essential roles in cell growth and drug response.

overall_design: To investigate the function of the 2 top hits from the genome-wide ultra-conserved elements (UCE) genetic screen by the established dual-CRISPR system, we first generated the homologous ultra-conserved element deleted cell lines using our dual-CRISPR system. We then performed the gene expression analysis from the total RNA-seq of the different UCE-KO cell lines. By comparative gene expression profiling analysis using DESeq2, we identified the differentially expressed genes between the wild-type K562 cells and 2 derivatives UCE-KO cell lines.

first: Baoxu

last: Pang

first: Yufeng

last: Li

first: Almira

last: Akkari-Henić


title: OsMADS6-OsMADS32 and REP1 control palea cellular heterogeneity and morphogenesis in rice

status: Public on Mar 09 2024

city: Shanghai

first: Xuelian

address: 800 Dongchuan Road

institute: Shanghai Jiao Tong University

summary: Precise regulation of cell proliferation and differentiation is a key factor for diversity in morphology during organogenesis. Rice palea comprises two distinct regions, the marginal region of palea (MRP) and the body of palea (BOP), which exhibit heterogeneity in cellular composition and behavior. This study reveals that REP1 promotes epidermal cell proliferation and differentiation in the BOP, forming hard silicified protrusion cells, by regulating the cyclin-dependent kinase gene, OsCDKB1;1. Conversely, OsMADS6 and OsMADS32 are expressed exclusively in the MRP, where they limit the division rates of epidermal cells by inhibiting OsCDKB2;1 gene expression, promoting endoreduplication and resulting in the formation of elongated epidermal cells. Furthermore, the OsMADS6-OsMADS32 complex and REP1 exhibit reciprocal inhibition to fine-tune the balance between cell division and differentiation during palea morphogenesis. Therefore, our study highlights the importance of controlling cellular heterogeneity in shaping plant morphological diversity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Osmads6, Osmads32, rep1 and wild type rice paleas

first: Qiang

last: Cai

first: Ling

first: Li


title: Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of embryonic stem cell maintenance and neuronal differentiation [RNA-seq]

status: Public on Mar 08 2024

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

institute: UCLA

summary: Maintenance of embryonic stem cells (ESCs) and their differentiation to neuronal lineages requires complex genetic regulation of transcription, splicing, and translation, but the interconnections of these processes is not well understood. We found that the splicing regulator polypyrimidine tract binding protein 1 (PTBP1) alters the neuronal transcriptional program. Transcripts for DPF2, a subunit of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, contain an additional exon (exon 7) in brain. This exon encodes a 14 amino acid insert in the C2H2-type zinc finger domain generating a longer DPF2-L isoform, compared to the canonical short DPF2-S isoform. We find that inclusion of exon 7 in Dpf2 mRNA is strongly inhibited by PTBP1, and loss PTBP1 switches expression from DPF2-S to DPF2-L early in neuronal differentiation. We created CRISPR/Cas9-mediated genome-edited cells that force expression of either DPF2-S or DPF2-L to examine the consequences of this switch in ESCs, in ESC-derived neural progenitor cells (NPCs), and in glutamatergic neurons (GNs). We found substantial changes in gene expression induced by DPF2-L in ESCs and by loss of this isoform in neurons. Chromatin immunoprecipitation in ESCs revealed that DPF2-S but not DPF2-L preferentially bound to genomic regions associated with the stem cell pluripotency transcription factors OCT4, SOX2, and NANOG. In NPC’s, DPF2-S preferentially bound to genomic regions co-occupied by the transcription factor NF1, while DPF2-L preferred sites bound by the CTC binding factor, CTCF. The correlation of DPF2 isoform binding with chromatin states identified by ChromHMM revealed that DPF2-S binds to sites enriched in enhancers, whereas DPF2-L-bound sites are enriched for promoters. Altogether, our study identifies a new neuronal BAF isoform that alters its functional targets, and demonstrates how splicing regulatory events during development elicit wide ranging impacts on chromatin organization and transcription.

overall_design: To investigate specific functions of alternative DPF2 isoforms in embryonic stem cells and during their differentiation to neurons, we engineered genome-edited mouse ESCs that force express either DPF2-S or DPF2-L. We have differentiated these cells to neural progenitors (NPCs) and glutamatergic neurons (GNs) and performed comperative gene expression profiling of RNA-seq data.

first: Mohammad

last: Nazim

first: Chia-Ho

first: Douglas

last: Black


title: FOXF1 Promotes Tumor Vessel Normalization and  Prevents Lung Cancer Progression through FZD4

city: Phoenix

first: Ying-Wei

last: Lan

address: 475 N. 5th Street

department: Child Health

institute: The University of Arizona College of Medicine-Phoenix

summary: Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific over-expression of FOXF1 normalized tumor vessels and inhibited progression of lung cancer. FOXF1-deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has a promise for future therapies in NSCLC.

overall_design: LLC tumors were micro-dissected from control (n = 4) and endFoxf1+/- (n = 4) mice 21 days post tumor cell injection. Endothelial cells (CD45-CD31+) cells were sorted from the micro-dissected lung tumors using FACS, and RNA was extracted and performed RNAseq.

first: Tanya

middle: V

last: Kalin

first: Guolun


title: Massively Parallel Reporter Assay Confirms Regulatory Potential of hQTLs and Reveals Important Variants in Lupus and Other Autoimmune Diseases

city: Oklahoma City

first: Patrick

last: Gaffney

state: OK

address: 825 NE 13th Street

department: Genes and Human Disease

institute: Oklahoma Medical Research Foundation

summary: We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on SLE and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele dependent manner. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared to other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants. In addition, we nominate 17 variants (including 11 previously unreported) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies primary and immortalized B cells.  Thus, we uncover important insights into the mechanistic relationships between genotype, epigenetics, and gene expression in SLE and AI disease phenotypes.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: A massivey parallel reporter assay to study the regulatory potential of histone QTLs and other variants relative to autoimmune disease; comparing MPRA in EBV cells from a SLE case (6 biological replicates) compared to plasma controls (4 biological replicates)

first: Yao

last: Fu

first: Jennifer

last: Kelly

first: Jaanam

last: Gopalakrishnan

first: Richard

middle: C

last: Pelikan

first: Kandice

last: Tessneer

first: Satish

last: Pasula

first: Kiely

last: Grundahl

first: David

last: Murphy


title: Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of embryonic stem cell maintenance and neuronal differentiation[ATAC-seq]

overall_design: To investigate chromatin accessibility across the genome in DPF2-S or DPF2-L expressing cells, we engineered genome-edited mouse ESCs that force express either DPF2-S or DPF2-L. We then performed transposase-accessible chromatin with sequencing assays (ATAC-Seq) in ESCs and ESC-derived NPCs.

first: An-Chieh

last: Feng


title: Allergen Challenge for Evoked Phenotypes in Asthma (the ACE Study)

first: Prescott

last: Woodruff

address: 513 Parnassus

department: DOM

institute: University of California San Francisco

summary: This protocol outlines a single-site mechanistic study aiming to investigate long RNAs differentially expressed in the airway epithelium of asthma patients both at baseline and in response to segmental airway allergen challenges. Over approximately 14 days, the study spanned three visits:  Visit 1: Comprehensive characterization of participants, encompassing lung function testing, methacholine challenge testing, and allergen skin prick testing.  Visit 2: Participants underwent bronchoscopy wherein three procedures were performed  a. Epithelial brushings were performed in a segmental airway (baseline sample)  b. Diluent (inactive control) was instilled into another segmental airway  c. A small dose of allergen was administered into a third segmental airway using standardized cat or dust mite allergen extracts.  Visit 3 (24 hours or 7 days post Visit 2): Another bronchoscopy was carried out to collect epithelial brushings in the diluent challenged and allergen challenged segments  The collected epithelial brush samples underwent analysis for mRNA expression in the epithelial brushings. The study successfully incorporated a total of 23 subjects, which included 18 asthmatics (with stable or well-controlled conditions), 2 allergic non-asthmatics, and 3 non-allergic non-asthmatics.

overall_design: This was a single-site, mechanistic study investigating the differential expression of long RNAs in the airway epithelium of asthma patients before and after allergen challenges. The study spanned approximately 14 days and consisted of three visits. Procedures include lung function tests, methacholine challenge tests, allergen skin prick tests, and bronchoscopies with segmental allergen administration. The allergens used were either cat or dust-mite standardized allergen extracts. The third visit involved a bronchoscopy to gather epithelial brushings, which were then analyzed to measure mRNA (long RNA) expression. The study cohort comprised 23 subjects, segmented into 18 asthmatics with stable or well-controlled asthma, 2 allergic non-asthmatics, and 3 non-allergic non-asthmatics.


title: ChIP-seq of colonic intestine epithelium from Vil-CreER;MTA2F/F (MTA2KO), Vil-CreER;SATB2F/F(SATB2KO) and Wild Type mice

first: Wei

last: Gu

state: Shanghai

address: 51 Rijing Road

department: Beigene Institute

institute: Beigene

summary: We use ChIP-seq to understand the genomic binding sites of MTA2 in colonic epithelium and to study the shifting of SATB2 and HNF4A's genomic binding sites after MTA2 loss in colonic epithelium.

overall_design: Intestine epithelium isolated from adult mice. Genotypes include Vil-CreER;MTA2F/F (MTA2KO) , Vil-CreER;SATB2F/F(SATB2KO) and MTA2f/f (Wild Type) mice.

first: Pratik

last: Singh

first: Ramesh

last: Shivdasani

first: Qiao

last: Zhou


title: Investigation of potential targets of TaSPL6-D in wheat

city: wuhan

first: yu

last: zhang

address: Gaonong International Phase II, No. 888 Gaoxin Avenue, Donghu High-tech Zone, Wuhan

institute: biorun

summary: Here, we performed the cleavage under targets and tagmentation (CUT&Tag) assay, a recently developed technology for protein-DNA interaction, in protoplasts extracted from wheat root tissues to genome-widely identity the potential targets of TaSPL6-D. Using CUT-Tag-seq, we obtained 7,403 confidential peaks from two biological replicates, with which 2688 genes was associated. Zooming in the promoter region, GTAC motif was significantly enriched in the peaks. Gene Ontology analysis of genes with binding peaks in the promoter region revealed that, the most enriched gene term is ‘response to temperature/heat stimuli’, which is consistent with previous studies on the functions of TaSPL6-D orthologs in rice and Arabidopsis. The following enriched terms are genes of ‘regulation of response to salt stress’, ‘regulation of response to osmotic stress’, and particularly ‘positive regulation of response to salt stress’, implying TaSPL6-D is an upstream regulator of salt stress response.

overall_design: DNA library established by Cleavage under targets and tagmentation (CUT&Tag) assay in wheat protoplasts was used for sequencing to genome-widely identity the potential targets of TaSPL6-D.

first: Meng

last: Wang

first: Weiming

last: Shi


title: Aging-induced tRNAGlu-derived fragment impairs glutamate biosynthesis by targeting mitochondrial translation-dependent cristae organization

city: Hefei

first: Ding

middle: feng

last: Li

address: Huang shan road 443

institute: USTC

type: Non-coding RNA profiling by high throughput sequencing

summary: We report the high-throughput sequencing of tsRNA expression in mouse brain

overall_design: Examination of tsRNA expression pattern in mouse brain

first: Qiang

last: Liu

first: Juan

last: Zhang

first: Dingfeng


title: Changes in Protein Fluxes in Skeletal Muscle During Sequential Stages of Muscle Regeneration After Acute Injury in Male Mice

city: Berkeley

first: Alec

last: Bizieff

address: 54 Mulford Hall

institute: UC Berkeley

summary: Background Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to pathology and clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Methods Here, we employed a workflow that couples deuterated water (2H2O) administration with tandem mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue. Results The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms. Conclusions In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets.

overall_design: Mice were randomly assigned to a group (n=5 per group) that reflected the stage of muscle regeneration being measured (Proliferation Stage, Differentiation Stage, Maturation Stage, or Uninjured Control). Cardiotoxin (CTX)-treated groups received a dose of 50 µL 0.1mg/mL CTX in sterile PBS/0.2% Meloxicam in the form of an intramuscular injection in the Tibialis Anterior (TA) muscle to one limb while the TA from the other limb was not injected. The day of CTX injection is called Day 0, and subsequent days allow for the toxin-induced muscle regeneration to occur. One group of mice did not receive CTX injection to serve as uninjured controls alongside experimental groups.

first: Maggie

last: Cheng

first: Kelvin

last: Chang

first: Marc

last: Hellerstein


title: Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine mediated regulation of c-Fos

city: Nanjing

first: Xi

address: Longmian Avenue 101, Jiangning District

institute: Nanjing Medical University

summary: CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME regulates Treg cell differentiation is not understood. Here we measured 630 metabolites in the TME and found that substrates required for the synthesis of sphingolipids, serine and palmitic acid, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos.  Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits anti-tumor immunity.

overall_design: To identify mechanisms via which sphinganine promoted the expression of FoxP3, we performed single cell RNA sequencing to analyze the transcriptional profiles of TCR-transgenic OT-II CD4+ T cells. To systematically study the influence of sphinganine on the chromatin accessibility and the binding of c-Fos, c-Jun, and NFAT2, we performed ATAC sequencing and CUT & RUN sequencing.

first: Sicong

last: Ma

first: Fuwei

last: Shang

first: Guoliang

last: Cui


title: PARP 1 inhibition mediates a switch from cell death to transient senescence improving repair from acute oxidative injury

status: Public on Mar 07 2024

city: GRONINGEN

first: Marco

last: Demaria

state: Groningen

address: Antonius Deusinglaan, 1

institute: umcg

summary: Excessive amounts of Reactive Oxygen Species (ROS) lead to macromolecular damage and high levels of cell death and pathological sequelae. Switching cell death to a tissue regenerativestate can potentially improve short – and long-term consequences of ROS-associated acute tissueinjury. However, the mechanisms regulating oxidative stress-induced cell fate decision and their manipulation for improving repair remain poorly understood. Here, we show that cells exposed tohigh oxidative stress enter a PARP1-mediated regulated cell death, and that blocking PARP1activation promotes conversion of cell death into senescence (CODIS). We demonstrate that CODIS depends on reducing mitochondrial Ca2+ overload as a consequence of retaining thehexokinase HKII onto mitochondria. In a mouse model of kidney ischemia/reperfusion damage,PARP1 inhibition lowers necrosis and increases acute and transient senescence at the injury site,leading to improved recovery from damage. For the first time, we provide evidence that strategiesconverting cell death into acute senescence can therapeutically reduce the detriment of accidental tissue damage. _x000B_

overall_design: In this study, we showed that PARP inhibition (using PJ34) has a protective effect on tissue integrity and cellularity in the short term (3 days after IR). To evaluate the long-term effect, mice were divided in different groups : 2 Control groups which have received either vehicle (NV) or the PARP inhibitor PJ34 (NP) and 2 groups that have underwent ischemia reperfusion (IR) surgery, where one group received vehicle (IRV), and the other group received PJ34 (IRP).

first: Jamil

last: Nehme

first: Abdullah

last: Altulea

first: Yao

last: lin


title: Analysis of human neuronal cells carrying ASTN2 deletion: A cross-disorder risk variant of psychiatric disorders

city: Nagoya

first: Yu

last: Hayashi

state: Aichi

address: 65 Tsurumai-cho, Showa-ku

department: psychiatry

institute: Nagoya University

summary: Recent genetic studies have found common genomic risk variants among psychiatric disorders, strongly suggesting the overlaps in their molecular and cellular mechanism. Our research group identified the variant in ASTN2 as one of the candidate risk factors across these psychiatric disorders by whole-genome copy number variation analysis. However, the alterations in the human neuronal cells resulting from ASTN2 variants identified in patients remain unknown. To address this, we used patient-derived and genome-edited iPS cells with ASTN2 deletion; cells were further differentiated into neuronal cells. A comprehensive gene expression analysis using genome-edited iPSC cells with the loss of function variants on both alleles revealed that the expression level of ZNF558, a gene specifically expressed in human forebrain neural progenitor cells, was greatly reduced in ASTN2-deleted neuronal cells.

overall_design: A total of 6 iPS cell lines, including healthy control (N=3) and homozygous ASTN2 deletion (N=3), were induced to neurosphere, respectively. Each of the healthy control and homozygous ASTN2 deletion iPS cell lines is the same clone and has a different number of passages.

first: Yuko

last: Arioka


title: YvcL-regulated genes in Enterococcus faecalis

city: MILWAUKEE

first: CHRIS

last: KRISTICH

address: 8701 WATERTOWN PLANK RD

institute: MCW

summary: The goal was to identify genes regulated by YvcL (EF0768) by assessing changes in the yvcL deletion mutant relative to the isogenic wild-type. Wild-type E. faecalis OG1 and the mutant lacking yvcL were cultured in Mueller-Hinton broth to exponential phase. Total RNA was purified, depleted for rRNA, and subjected to preparation of mRNA libraries.

overall_design: Four biological replicates of each strain (wild-type, yvcL deletion mutant) were prepared. Cultures of the strains were grown at 37 C in Mueller-Hinton broth until exponential phase prior to collection and RNA extraction.

first: Samantha

middle: N

last: Atkinson

first: Dusanka

last: Djoric

first: Christopher

last: Kristich


title: Novel Biomarkers and Interferon Signature in Secondary Progressive Multiple Sclerosis

city: Chicago

first: Anthony

last: Reder

state: IL

address: 5841 S Maryland Ave

department: Neurology

institute: University of Chicago Medicine

summary:
Transcriptome analysis of RNA samples from human PBMCs of IFN-beta-1a (Rebif) therapy in secondary progressive multiple sclerosis (SPMS) patients.
Objective: Untreated multiple sclerosis is inflammatory, with decreased immune control and subnormal type I interferon (IFN) signaling. IFN-ß therapy corrects abnormal IFN-ß signaling, reduces inflammation on MRI, exacerbations, and disease worsening in relapsing-remitting MS (RRMS). For unclear reasons, secondary progressive MS (SPMS) exhibits waning attacks, relentless neurodegeneration, and diminished benefits of therapy.     Methods: Peripheral blood mononuclear cells and serum were from 10 healthy controls (HC), 9 therapy-naive RRMS, 20 therapy-naïve SPMS, and 10 IFN-ß-treated SPMS patients after therapy washout and four hours after IFN-ß-1a injection. Global gene expression was assayed with sensitive RNA microarrays and multiplexed serum immune and neuroprotective protein assays.    Results: Therapy-naive RRMS cells displayed 8,723 differentially expressed genes (DEG), compared to HC, vs. d only 3,936 DEG in therapy-naive SPMS. In SPMS, gene expression was subnormal in the WNT/ß-catenin pathway that suppresses inflammation and enhances blood-brain barrier integrity. Olfactory receptor (OR) genes, linked to lymphocyte migration, were overexpressed in RRMS (111 DEG), intermediate in SPMS (34 DEG) vs. HC, differentiating RRMS from SPMS (p < 0.007). IFN-ß injections in SPMS decreased expression of pro-inflammatory genes and increased anti-inflammatory, anti-oxidant metallothionein genes. Pro-inflammatory and anti-inflammatory protein levels were balanced in HC, disrupted in RRMS, and intermediate in SPMS.     Interpretation: Aberrant gene expression seen in RRMS wanes in SPMS, paralleling fewer clinical exacerbations and diminished therapeutic responses. Novel biomarkers for SPMS suggest new targets to correct subnormal immune regulation and brain repair in this neurodegenerative disease.

overall_design: 59 blood and serum samples were collected from 49 subjects, including 10 healthy controls (HC), 9 untreated clinically stable RRMS, and 20 untreated SPMS (10 slowly progressive, 10 rapidly progressive). 10 long-term subcutaneous IFN-ß-1a-treated SPMS (Rebif, 44 mcg thrice weekly) provided paired samples after > 60-hour IFN washout (73.0 ± 8.9 hours; mean ± SEM) and four hours after IFN injection (4.07 ± 0.03 hours). Rapid, short-term RNA induction was studied 4 hours after injections, because injected IFN-beta levels peak at 30 minutes, followed by expression of many IFN-induced genes (ISG) within 2-3 hours. These include cytokines and also multiple subtypes of IFN-beta and IFN-alpha, based on serum IFN activity assays

first: Avital

last: Fogel

first: Maya

last: Olcer

first: Aika

last: Goel

first: Xuan

last: Feng

middle: T


title: Comparable CD8 + T cell responses to SARS-CoV-2 vaccination in single-cell transcriptomics of recently allogeneic transplanted patients and healthy individuals

city: Berlin

first: Benedikt

last: Obermayer

address: Chariteplatz 1

department: Core Unit Bioinformatics

institute: Berlin Institute of Health

summary: Despite extensive research on SARS-CoV-2 vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T-cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT). In this study, we assessed the serological response and performed longitudinal multimodal analyses including T cell functionality and single-cell RNA sequencing combined with TCR/BCR profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees. Protective antibody titers were achieved in 40% of patients. We identified a distorted B and T cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. IGHV gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRβ gene rearrangement after vaccination differed from patterns observed in healthy vaccinees as well as unvaccinated ASCT patients and associated transplant donors. Crucially, ASCT patients elicited comparable proportions of SARS-CoV-2 vaccine-induced (VI) CD8+ T cells, characterized by a distinct gene expression pattern that is associated with SARS-CoV-2 specificity in healthy individuals. Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VI-CD8+ T cells can be induced in part of ASCT patients, our data advocate early post-transplant vaccination due to the high risk of infection in this vulnerable group.

overall_design:
Peripheral blood from patients <6 months after ASCT was collected before the first, after the first and after the second vaccine shot with BNT162b2 during 06/2021-08/2022. Serology, T cellular response as well as single-cell RNA sequencing combined with TCR/BCR profiling was conducted in order to dissect immune response to vaccinations and  unravel the peculiarities of the vaccination response after ASCT.

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Eva

last: Tranter

first: Il-Kang


title: Analysis of mRNA expression of livers from (PR)97 expressing mice

first: Orlando

last: Domínguez

address: Melchor F. Almagro 3

institute: CNIO

summary: Nucleolar stress (NS) kills cells by p53-dependent and independent manners. To investigate the mechanisms of p53-indendendent toxicity, we here used (PR)n arginine-rich peptides as inducers of NS, which are found in patients of some neurodegenerative diseases. Although these peptides generate NS, how this translates to toxicity is poorly understood. We here reveal that whereas (PR)n expression leads to an overall decrease in protein abundance for the majority of the proteome, this occurs concomitant to an accumulation of free ribosomal proteins in the cytoplasm, which is a hallmark of ribosomopathies. Conversely, cells with acquired resistance to (PR)n peptides present a global downregulation of ribosomal proteins and low levels of mTOR signaling. In mice, systemic expression of (PR)97 drives widespread NS and accelerated ageing, associated to an increased expression of ribosomal proteins and mTOR hyperactivation. Furthermore, the progeroid phenotype of (PR)97-expressing mice is alleviated by rapamycin. Importantly, we show that the generalized accumulation of free ribosomal proteins is not restricted to (PR)n peptides, but is a common outcome in response to chemical or genetic perturbations that generate NS such as Actinomycin D, TIF-IA depletion or the expression or mutant HMGB1 forms recently associated to rare human diseases. Together, our study provides in vivo evidence for the role of NS as a driver of ageing in mammals, and describes a general model to understand the mechanisms behind p53-independent cell toxicity caused by NS.

overall_design: We used liver samples from mice with inducible (PR)97 expression, and non-inducible controls. Total RNA was extracted from liver tissue of non-inducible (Col1A1PR97/+ Rosa26+/+ (PR+/+)) and inducible ((Col1A1PR97/+ Rosa26rtTA/+ (PR+/KI)))  male litermates after 17 weeks’ treatment with oral doxycycline.

first: Oleksandra

last: Sirozh

first: Vanesa

last: Lafarga

first: Oscar

last: Fernandez-Capetillo