title: Transcriptomic profiling of human pancreatic cancer-derived cell lines  PANC-1 depleted of TRIP12 mRNA compared to control cells

first: jerome

last: torrisani

address: 2 avenue Hubert Curien

institute: inserm

summary:
TRIP12 (Thyroid hormone Receptor Interacting Protein) is an E3 ubiquitin ligase involved in numerous cellular processes. TRIP12 controls the stability of the PTF1A transcription factor that is implicated in pancreatic cancer initiation.
To elucidate the role of TRIP12 on the expression of pancreatic cancer-derived cell lines, Trip12 mRNA expression was inhibited using a shRNA strategy. Transcriptomic profiling of shTrip12-PANC-1 cell s was perfomed and compared to ShScramble control cells.

overall_design: Two-condition experiment. PANC-1 shTRIP12 vs PANC-1 ShControl. Biological replicates: 4 Shcontrol replicates, 4 ShTRIP12 replicates.

first: Jerome

last: Torrisani

first: Lucrece

last: Dagnon

first: Pascal

middle: G

last: Martin

first: Yannick

last: Lippi


title: Transcriptomic profiling of human pancreatic cancer-derived cell lines  MiaPACA-2 depleted of TRIP12 mRNA compared to control cells

status: Public on Apr 19 2024

city: Toulouse

country: France

type: Expression profiling by high throughput sequencing

summary:
TRIP12 (Thyroid hormone Receptor Interacting Protein) is an E3 ubiquitin ligase involved in numerous cellular processes. TRIP12 controls the stability of the PTF1A transcription factor that is implicated in pancreatic cancer initiation.
To elucidate the role of TRIP12 on the expression of pancreatic cancer-derived cell lines, Trip12 mRNA expression was inhibited using a shRNA strategy. Transcriptomic profiling of shTrip12-MiaPACA-2 cell s was perfomed and compared to ShScramble control cells.

overall_design: Two-condition experiment. MiaPACA-2 shTRIP12 vs MiaPACA-2 ShControl. Biological replicates: 4 Shcontrol replicates, 4 ShTRIP12 replicates.


title: UV- and g-radiation induced molecular changes for rapid lipid accumulation in Chlorella sorokiniana

city: Mumbai

first: Dr. Ashish Kumar

last: Srivastava

state: Maharashtra

address: Anushakti Nagar, Trombay

country: India

institute: Bhabha Atomic Research Centre

summary: The data in the present study suggested the possibility of using UV and γ-radiation to enhance lipid accumulation along with detailed insights at the molecular level.The transcriptomic data at 24 h post UV (1 J/cm2) and γ-radiation (1 kGy) revealed foremost effect of UV on photosynthetic machinery while in γ-, DNA damage, repair and replication at a global level. In addition, the key fatty acid biosynthesis related genes were also found to be upregulated which coincided with 1.56- and 1.73-fold increase in lipid content

overall_design: Change in mRNA profile of Chlorella sorokiniana in response to UV and gamma with respect to control sample 24h post irradiation.

first: Devi

middle: R

last: Singh Reema

first: Ashish

middle: K


title: RNAseq data for FACS sorted glial cells KPNA4 KO mice for Importin α4 deficiency induces psychiatric disorder-related behavioral deficits and neuroinflammation in mice.

status: Public on Apr 18 2024

city: Suita

first: Koki

last: Sakurai

state: Osaka

address: Yamadaoka 3-2

department: Institute for Protein Research

country: Japan

institute: Osaka University

summary: Importin α4, which is encoded by the Kpna4 gene, is a well characterized nuclear-cytoplasmic transport factor known to mediate transport of transcription factors including NF-κB. Here, we report that Kpna4 knock-out (KO) mice exhibit psychiatric disorder-related behavioral abnormalities such as anxiety-related behaviors, deceased social interaction and sensorimotor gating deficits. Contrary to a previous study predicting attenuated NF-κB activity as a result of Kpna4 deficiency, we observed a significant increase in expression levels of NF-κB genes and pro-inflammatory cytokines such as TNFα, Il1β or Il-6 in the Prefrontal Cortex or Basolateral Amygdala of the KO mice. Moreover, examination of inflammatory responses in primary cells revealed that Kpna4 deficient cells have an increased inflammatory response, which was rescued by addition of not only full-length, but also a nuclear transport deficient truncation mutant of importin α4, suggesting contribution of its non-transport functions. Furthermore, RNAseq of sorted adult Microglia and Astrocytes and subsequent transcription factor analysis suggested increases in Polycomb repressor complex 2 (PRC2) activity in Kpna4 KO cells. Taken together, importin α4 deficiency induces psychiatric disorder-related behavioral deficits in mice, along with an increased inflammatory response and possible alteration of PRC2 activity in glial cells.

overall_design:
To investigate the molecular alterations behind the unexpected increase in proinflammatory activation induced by Kpna4 deficiency, we examined individual glial cell types to understand gene expression profiles in response to a proinflammatory stimulus. We applied a tissue disassociation-cell sorting strategy utilizing florescence activated cell sorting (FACS), to isolate both microglial (MG) and AST populations from mice administered LPS to stimulate inflammation.
20 hours before dissection, 1mg,/kg of LPS (L2880; Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally injected into 8 to 9 week-old WT and KPNA4 KO mice as an inflammatory stimulus. Animals were deeply anesthetized with isoflurane and used for subsequent cell collection and FACS experiments

first: Yoshiatsu

last: Aomine

first: Takatoshi

last: Hikida

first: Yoichi

last: Miyamoto


title: Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing

city: Seattle

first: Daniel

last: Zak

state: WA

address: 188 East Blaine Street, Suite 300

country: USA

institute: Regeneron

summary: Chimeric antigen receptor designs that incorporate pharmacologic control are desirable, however designs suitable for clinical translation are needed. We designed a fully human, rapamycin regulated, drug product for targeting CD33+ tumors called dimerization agent regulated immunoreceptor complex (DARIC33). T cell products demonstrated target specific and rapamycin dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following re-exposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for OFF-ON state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase 1 DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and anti-tumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

overall_design:
DARIC33 cells and untransduced T cells derived from n = 4 healthy donors were incubated with 1nM rapamycin or media alone prior to culture alone or with CD33+ MV4-11 AML target cells. Following coculture, CD4+ and CD8+ cells were sorted and evaluated by RNA-Seq.
This experiment compares transcriptomes of untransduced and DARIC33 transduced CD4+ and CD8+ T cells that were derived from four donors after 24hr incubation across two variables: with or without 1nM rapamycin and with or without activation by CD33+ MV4-11 AML target cells.  In addition, transcriptomes were assessed at the 168hr timepoint for CD4+ and CD8+ DARIC33 transduced T cells derived from 3 donors and incubated with 1nM rapamycin and activated by CD33+ MV4-11 AML target cells.

first: Jacob

last: Appelbaum

first: April

last: Price

first: Alexander

last: Astrakhan

first: Michael

last: Jensen

middle: E


title: Cranial photo-immunologic regulation along the skull-meninges-brain axis for promoting recovery from ischemic brain injury

city: Zhanjiang

first: Wei

last: Feng

address: 236 Yuanzhu Road

country: China

institute: Central People's Hospital of Zhanjiang

summary: Stroke involves in the interaction between central and peripheral immune systems. Skull bone marrows serve as reservoirs for immune cells in brain borders, and can rapidly respond to perturbations in the brain environment. Hence, targeting the skull bone marrow to modulate neuroimmune communications along the calvaria-meninges-brain axis would potentially improve stroke prognosis. Here, we successfully achieved cranial immunomodulation via ultraviolet (UV) irradiation of the interparietal region, which was characterized by rich marrow cavities and channels connecting the skull and meninges. Utilizing the recently-developed long-term clearing cranial window that ensured the integrity of skull, we discovered that the cranial photo-immunologic regulation (CPR) could promote cerebrovascular regeneration and aid in neurovascular repair post ischemic stroke. Single-cell transcriptome analysis revealed that meninge could be a crucial neuroimmune interface for ischemic stroke-induced immune responses. And, CPR could restore the stroke-induced alterations in cellular gene expression, especially meningeal B cells. Further we demonstrated that CPR could effectively alleviate the excessive suppression of meningeal B cell activation caused by ischemic stroke. This work opens avenues for immunoregulation through the skull-meninges-brain axis and provides valuable insights for immunomodulatory therapies in brain diseases.

overall_design: Bulk RNA sequencing of mouse skull, meninges, and brain tissue at 0.5/24 hours post cranial photobiomodulation, as well as bulk RNA sequencing of mouse skull, meninges, and brain tissue following non-cranial  photobiomodulation.

first: Chao

last: Zhang

first: Chun-Jie

last: Liu

first: Jianhao

last: Zeng

first: Lisi

last: Wang

first: Xin

last: Dai

first: Tao

last: Tao


title: RNA-seq of E0771-Vector control, E0771-Malt1-WT  and E0771 Malt1-PD (Without T cell coculture)

city: Beijing

first: Hanqiu

last: Zheng

address: Haidian District

institute: Tsinghua university

summary: To reveal the downstream paracrine signals from WT-Matl1 or PD-Malt1 tumor cells which responsible for the above observed macrophage phenotypes, we collected mRNA samples from Vector control-, WT-Malt1-, PD-Malt1-, V87R-Malt1-, or L88D-Malt1-expressing E0771 cancer cells, and performed RNA-seq analysis.

overall_design: E0771 cells expressing either -Vector control, wild type-Malt1 (M-WT), Malt1 PD mutant (M-PD), Malt1 V87R and Malt1 L88D were lysed by TRIZOL for RNA extraction,  perform reverse transcription and cDNA library for RNA-Seq analysis.

first: Yuwei

first: Chen

last: Tian


title: Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance II

city: Bethesda

middle: C

last: Kelly

state: MD

address: 41 Center Drive

department: Center for Cancer Research

institute: NCI

summary: Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations; namely, Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21 that attracts developing thymocytes to the medullary region. Here we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detected a cooperation for self-tolerance between Aire and Fezf2, which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

overall_design: Single Cell 3' end-counting gene expression (scRNA-Seq) using the 10X Genomics platform to analyze thymic epithelial cells from genetically engineered mice.

first: Yousuke

last: Takahama

first: Naozumi

last: Ishimaru

first: Aya

last: Ushio

first: Mami

last: Matsuda-Lennikov

first: Felix

last: Kalle-Youngoue

first: Akihide

last: Shimizu

first: Abdallah

last: Abdelmaksoud


first: Wei

last: Feng

overall_design: Single-cell transcriptomic profiling was performed on Sham, tMCAO (transient middle cerebral artery occlusion), and tMCAO+UVB(ultraviolet B) treated mouse skull, meninges, and brain tissue separately.


title: Gene expression profile at single cell level of CD45+ immune cells from E0771-Vector control tumor tissue, E0771 wildtype Malt1 overexpression tumor tissue and E0771 paracaspase-deficient Malt1 overexpression tumor tissue.

summary: To invesitigate the additional paracaspase-independent tumor-promoting effect exist for Malt1. We thus collected  E0771-Vector control tumor tissue, E0771 wildtype Malt1 (Malt1-WT) overexpression tumor tissue and E0771 paracaspase-deficient Malt1 (Malt1-PD) overexpression tumor tissue, and sorted out immune cells for 10× Genomics single cell RNA-sequencing (scRNA-seq).

overall_design: First inject E0771-Vector control,E0771 Malt1-WT overexpression, and E0771 Malt1-PD overexpression cells into the mammary fatty pad of C57B6/J mice to form tumor. We thus collected  E0771-Vector control tumor tissue, E0771 Malt1-WT overexpression tumor tissue and E0771 paracaspase-deficient Malt1 (Malt1-PD) overexpression tumor tissue, and sorted out immune cells by FACS for 10× Genomics single cell RNA-sequencing (scRNA-seq).


title: RNA-seq of E0771-Vector control, E0771-Malt1-WT  and E0771 Malt1-PD after T cell killing

summary: To understand potential downstream signaling molecules that are responsible for wild type-Malt1 induced resistance of tumor cell to CD8 T cell killing , we first use  E0771 cells expressing either -Vector control, wild type-Malt1 (M-WT), or Malt1 PD mutant (M-PD) to coculture with activated CD8 T cell at a effctor: target ratio(E:T ration) of 3 for 12 hours. Then wash the cells with PBS, digest the cells by 0.25% trypsin solution, harvest the cells in 15 ml tubes by centrifugation (1000 rpm 5min) and seed cells into the original plate with DMEM+10%FBS+1%P/S medium. 6 hours after sseding,  wash cells with PBS, lyse cells with TRIZOL for RNA extraction,  perform reverse transcription and cDNA library for RNA-Seq analysis.

overall_design: E0771 cells expressing either -Vector control, wild type-Malt1 (M-WT), or Malt1 PD mutant (M-PD) were co-cultured with activated CD8 T cell at a effctor: target ratio (E:T ration) of 3 for 12 hours. Then wash the cells with PBS, digest the cells by 0.25% trypsin solution, harvest the cells in 15 ml tubes by centrifugation (1000 rpm 5min) and seed cells into the original plate with DMEM+10%FBS+1%P/S medium. 6 hours after sseding,  wash cells with PBS, lyse cells with TRIZOL for RNA extraction,  perform reverse transcription and cDNA library for RNA-Seq analysis.

first: Tengjiang

last: Zhang


title: Mutations in the splicing factor SF3B1 are linked to frequent emergence of HLA-DRlow/neg monocytes in lower-risk myelodysplastic neoplasms

city: Dresden

first: Susann

last: Winter

address: Fetscherstrasse 74

country: Germany

institute: University Hospital Carl Gustav Carus, Faculty of Medicine Carl Gustav Carus, TU Dresden

summary: Recurrent mutations in splicing factors, in particular the splicing factor 3B subunit 1 (SF3B1), are commonly seen in lower-risk (LR) myelodysplastic neoplasms (MDS) and result in various aberrantly spliced transcripts. The cell type-specific changes and their contribution to immune dysregulation in MDS remain vaguely understood. In this study, we performed RNA sequencing on classical monocytes isolated from patients with an isolated SF3B1K700E hotspot mutation. Our analysis revealed downregulated expression of genes involved in inflammatory cytokine signaling, as well as differential mRNA splicing of several genes involved in the regulation of the defense response and cytokine signaling, mRNA metabolism, apoptotic signaling, and mitotic cell cycle.

overall_design: Classical monocytes (CD14+CD16-) were magnetically isolated from viably frozen peripheral blood mononuclear cells (PBMCs) of treatment-naïve LR-MDS patients (n = 3 SF3B1K700E, n = 3 SF3B1wt) and hematologically healthy donors (HD, n = 3) as reference, yielding routinely >95% CD14+CD16- monocytes based on flow cytometric assessment. Monocytes were cultured/rested for 4 hours (195000 – 500000 cells per 96-well in 250 µl RPMI 1640/PenStrep/5% heat-inactivated human serum), followed by RNA isolation with TRIzol and Direct-zol™ RNA Microprep, mRNA library preparations, and sequencing reactions.

first: Marie

last: Schneider

first: Rita

last: Antunes Dos Reis

first: Desmond

last: Choy

first: Uwe

last: Platzbecker

first: Shahram

last: Kordasti


title: Effects of inhibitors in mice with oxygen-induced retinopathy

city: Fukui

first: Kengo

last: Nakanishi

address: 3-9-1 Bunkyo,Fukui

department: Applied Chemistry & Biotechnology

institute: University of Fukui

summary:
Analysis of gene expression changes in oxygen-induced retinopathy mice treated intravitreally with inhibitors.
oxygen-induced retinopathy(OIR)mice  is one of the experimental systems that mimic retinal ischemic diseases. This model is widely used as an evaluation system for various surgical procedures.

overall_design: Genes that increased in oxygen-induced retinopathy(OIR) and reverted to their original state with ISOX DUAL were extracted.

first: Makoto

last: Gozawa

first: Yoshihiro

last: Takamura

first: Masaru

last: Inatani

first: Masaya

last: Oki


title: Effect of a synthetic steroid 5α-androst-3β, 5α, 6β-triol on gene expression in irradiated BV2 cells

city: Guangzhou

first: Yamei

last: Tang

state: Guangdong

address: 107 Yan Jiang West Road

department: Department of Neurology

institute: Sun Yat-Sen Memorial Hospital

summary: We aimed to study the potential role and underlying mechanism of 5α-androst-3β, 5α, 6β-triol on irradiated BV2 cells. We established radiation induced BV2 cells injury model and  pretreated with 5α-androst-3β, 5α, 6β-triol  or vehicle. We then performed gene expression profiling analysis using data obtained from RNA-seq of different groups.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CV: Control+vehicle (C), RV: Radiation+vehicle (R), RD: Radiation+5α-androst-3β, 5α, 6β-triol (Y) groups

first: Ke

last: Zhong

first: Yu

last: Song

first: Sitai

last: Chen


title: Set7 deletion attenuates isoproterenol-induced cardiac fibrosis and prevents heart failure

city: Los Angeles

first: Yao Wei

last: Lu

state: CA

address: 2011 Zonal Ave

department: Department of Medicine

institute: University of Southern California

summary: The Set7 methyltransferase regulates the expression of several genes through the methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in cardiac remodeling and heart failure remains unknown. To address this question, wild type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol (60 mg/kg) or saline subcutaneously for 14 days. WT mice injected with isoproterenol displayed a decrease in Set7 activity in the heart, although Set7 protein levels were unchanged. WT and Set7 KO mice injected with isoproterenol exhibited an increase in the heart weight and cardiomyocyte area compared to their respective controls. However, Set7 KO mice displayed an exacerbated cardiac hypertrophy in response to isoproterenol compared to WT mice. In addition, Set7 deletion attenuated isoproterenol-induced cardiac fibrosis. Echocardiograms revealed that WT mice injected with isoproterenol had lowered ejection fractions and fractional shortening, and increased E/A ratios compared to their controls. Conversely, Set7 KO mice did not show alteration in these parameters in response to isoproterenol. Both isoproterenol and Set7 deletion changed the transcriptional profile of the heart. Moreover, Set7 deletion increased the expression of mitochondrial biogenesis and antioxidant factors in the heart and reduced the expression of cellular senescence and inflammation markers. Taken together, our data suggest that Set7 deletion reduces isoproterenol-induced myocardial fibrosis and prevents heart failure, suggesting that Set7 plays an important role in cardiac remodeling and function.

overall_design: The global Set7 knockout (KO) mice were generated by heterozygous crossings. All animals were treated with saline or isoproterenol (60mg/kg subcutaneously) for 14 consecutive days prior to examination and collection.

first: Guilherme

last: Lunardon

first: Tábatha

last: de Oliveira Silva

first: Gabriela

middle: P

last: Diniz


title: The Rapid Evolution of Chronic Inflammation to Tolerance / Immune Suppression: Mapping Bidirectional Checkpoints

status: Public on Apr 17 2024

city: Tallahassee

first: Elizabeth

last: Mazzio

state: Florida

address: College of Pharmacy and Pharmaceutical Sciences, 241 Fred Humphries Science Research Facility, 1515 MLK Blvd, Tallahassee , Florida 32307

institute: FAMU College of Pharmacy

summary: Currently, there is truly a universal lack of knowledge as to exactly how chronic inflammation leads to the initiation of cancer. Likewise, there exists an equal deficiency in knowledge to define the phenomenon of immune “ tolerance”. Tolerance is, however, generally described as the diminished biological response to repeated or sustained antigen exposure associated with limitation to efficacy of toll-like receptor (TLR) synthetic lipid A lipoglycans, or microbial antigen molecular pattern (MAMP) based tumor immune therapies. The current work was designed to gain greater understanding into these questions, by employing a model of sustained antigenic TLR4 activation with gram-negative cell wall (LPS) for up to 11 days in macrophages under highly controlled conditions.

overall_design: Raw 264.7 macrophages were treated with 500ng/ml for up to 11 days.  Phenotype evolution was monitored by WT RNA sequencing & protein spot check for: baseline (resting), 1-day LPS (acute), 7- & 11-day LPS (sustained chronic).Long term chronic inflammation required 18 hour periodic treatment changes, high volume/cell density ratio to ensure removal of cellular by-products that could create interfering (false) artifacts.

first: Andrew

last: Barnes


title: Effects of strigolactones on tomato leaf transcriptome under irrigated and repeated water stress conditions

city: Grugliasco

first: IVAN

last: VISENTIN

state: Seleziona uno Stato

address: Largo Braccini 2

department: DISAFA

country: Italy

institute: University of Turin

summary: In response to environmental stressors, plants exhibit a phenomenon known as stress memory, whereby a prior stress episode influences their subsequent responses. This suggests the existence of a molecular memory mechanism that allows plants to adapt to recurring stress events. Previous work has highlighted the contribution of the phytohormone strigolactones to drought stress and memory thereof (10.1007/s00425-015-2266-8; 10.1111/nph.14190; 10.1111/pce.13758; 10.1093/pcp/pcac058). Additionally, there are sparse indications that especially (but not only) in Solanaceous plants, they may have a significant effect on reproduction (reviewed by 10.1111/pce.14461). However, the molecular underpinnings of both phenomena have not been researched yet. This study investigated the positioning of strigolactones to the tomato flowering network, and their contribution to drought memory.

overall_design: We deep-sequenced total RNA from leaves of 3-week-old wt (M82 genotype) and strigolactone-depleted plants showing 70-80% reduction of strigolactone content in root tissues and exudates (SL-;  line 6936 silenced in the CCD7 biosynthetic gene, kind gift by Dr. H. J. Klee, University of Florida, ). A total of 21 samples are included in the dataset, corresponding to 7 different conditions/treatments/genotypes each coming in 3 biological replicates; each biological replicate consisted of at least 3 fully expanded leaves (one per plant) from 5 plants per condition/treatment/genotype. The 7 kinds of samples are leaves from plants under: unstressed, irrigated conditions (control samples, both wt and SL- genotype - WT_WW and CC_WW); irrigated conditions after 1 drought episode (recovery 1, both for wt and SL- genotype - WT_S1 and CC_S1); irrigated conditions after 2 drought episodes ( (recovery 2, both for wt and SL- genotype - WT_S2 and CC_S2); unstressed, irrigated conditions but treated 24 h earlier with the synthetic strigolactone analogue GR245DS (wt only - WTTR24).

first: Ivan

last: Visentin

first: Giulia

last: Russo

first: Caterina

last: Capezzali

first: Paolo

last: Korwin Krukowski

first: Eva

last: Campo

first: Andrea

last: Schubert

first: Francesca

last: Cardinale


title: Post-resolution macrophage-derived lipids shapes long-term tissue immunity and integrity

city: London

first: Derek

middle: W

last: Gilroy

address: 5 University Street

country: United Kingdom

institute: UCL

summary: Resolving inflammation is thought to return the affected tissue back to homeostasis. However, we show that within days following resolution of Streptococcus pneumonia-triggered lung inflammation there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing an alveolar or interstitial phenotype. Transcriptomic analysis revealed that macrophages are enriched with genes that drive T cell chemotaxis and differentiation as well as prostaglandin biosynthesis. Therapeutic depletion of post-resolution macrophages, inhibition of PGE2 synthesis or antagonism of EP4 reduced numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the a-integrin, CD103. This intervention resulted in a failure of these cells to reappear and reactivate upon secondary challenge up to six weeks following primary infection. Concomitantly, EP4 antagonism caused accumulation of lung macrophages and marked tissue fibrosis. Hence, resolving inflammation triggers a second wave of immune activity controlled, in part, by PGE2, which through EP4, drives local tissue-resident T cell development on the one hand, whilst limiting tissue injury on the other

overall_design:
To understand post-resolution mononuclear phagocyte function and the immuno-biology they control, macrophages bearing an alveolar phenotype as well as the three populations of interstitial macrophages
Three populations of interstitial macrophages (Figure 2E-H) were sorted using fluorescence-activated single cell sorting (FACS) from naïve as well as day 14 post S. pneumonia challenged mice and their transcriptomes sequenced using Illumina NovaSeq.

first: Karen

middle: T

last: Feehan

first: Hannah

last: Bridgewater

first: Jan

last: Stenkiewicz-Witeska

first: Roel

last: De Maeyer

first: John

last: Ferguson

first: Matthias

last: Mack

first: Jemery

last: Brown

first: Giuseppe

last: Ercoli

first: Connar

middle: M

last: Mawer

first: Arne

middle: N

last: Akbar

first: James

last: Glanville

first: Parinaaz

last: Jalali

first: Olivia

middle: V

last: Bracken

first: Anna

last: Nicolaou

first: Alexandra

last: Kendall

first: Michelle

middle: A

last: Sugimoto

last: Gilory


title: Shared Transcriptomic Signatures of Inflammaging Among Diverse Strains of Drosophila melanogaster

city: Gainesville

first: Lei

last: Zhou

state: FL

department: Molecular Genetics and Microbiology

institute: University of Florida

summary: Background: A prominent hallmark of aging is inflammaging—the increased expression of innate immune genes without identifiable infection. Model organisms with shorter lifespans, such as the fruit fly, provide an essential platform for probing the mechanisms of inflammaging. Multiple groups have reported that, like mammalian models, old flies have significantly higher levels of expression of anti-microbial peptide genes. However, whether some of these genes—or any others—can serve as reliable markers for assessing and comparing inflammaging in different strains remains unclear. Methods and Results: We compared RNA-Seq datasets generated by different groups. Although the fly strains used in these studies differ significantly, we found that they share a core group of genes with strong aging-associated expression. In addition to anti-microbial peptide genes, we identified other genes that have prominently increased expression in old flies, especially SPH93. We further showed that machine learning models can be used to predict the “inflammatory age” of the fruit fly.

overall_design: Fruit flies were aged in 25 to designated age and processed for RNA extraction.

first: Sabrina

middle: I

last: Perna

first: Weihao

last: Tang

first: Andrew

last: Li

first: Sydney

middle: D

last: Blimbaum


title: Porphyrin overdrive rewires cancer cell metabolism

city: Tampa

first: Rays HY

last: Jiang

address: 3720 Spectrum Blvd, Suite 304

department: Department of Global Health

institute: University of South Florida

summary: All cancer cells reprogram metabolism to support aberrant growth. Here, we report that cancer cells employ and depend on imbalanced and dynamic heme metabolic pathways, to accumulate heme intermediates, i.e., porphyrins. We coined this essential metabolic rewiring ‘porphyrin overdrive’ and determined that it is cancer-essential and cancer-specific. Among the major drivers are genes encoding mid-step enzymes governing the production of heme intermediates. CRISPR/Cas9 editing to engineer leukemia cell lines with impaired heme biosynthetic steps confirmed our whole genomic data analyses that porphyrin overdrive is linked to oncogenic states and cellular differentiation. While porphyrin overdrive is absent in differentiated cells or somatic stem cells, it is present in patient-derived tumor progenitor cells, demonstrated by single cell RNAseq, and in early embryogenesis. In conclusion, we identified a dependence of cancer cells on non-homeostatic heme metabolism, and we targeted this cancer metabolic vulnerability with a novel “bait-and-kill” strategy to eradicate malignant cells.

overall_design: Blast cells from AML patient donors with over 60% blast expansion in marrow biopsies were isolated according to standard tissue-banking protocols, washed and resuspended at 10^6 cells/ml. Cells were processed using the 10x Genomics Chromium controller and loaded according to standard protocol of the Chromium single cell 3’ library and v3 gel bead kit to capture 2,000 cells/Chromium Chip B. Single-cell capture, cell lysis, reverse transcription, and library preparation were performed per manufacturer instructions. Sequencing was performed on Illumina NextSeq 550 targeting150,000 reads/cell. The Cell Ranger Single-Cell software Suite (10x Genomics) was used for data processing, sample demultiplexing and gene expression quantification. t-Distributed stochastic neighbor embedding (t-SNE) and k-means clustering were used to reduce the data to a two-dimensional space and identify cell populations. Once the mean expression profiles of all cells were calculated, each cell was assigned to a subpopulation by the highest Spearman’s correlation.

first: Swamy

last: Adapa

first: Rays

middle: H


title: Human long noncoding RNA, VILMIR, is induced by major respiratory viral infections and modulates the host interferon response

city: Raleigh

first: Xinxia

last: Peng

address: 1060 William Moore Drive

institute: NC State

type: Non-coding RNA profiling by high throughput sequencing

summary: Long noncoding RNAs (lncRNAs) are a newer class of noncoding transcripts identified as key regulators of biological processes. Here we aimed to identify novel lncRNA targets that play critical roles in major human respiratory viral infections by systematically mining large-scale transcriptomic datasets. Using bulk RNA-sequencing (RNA-seq) analysis, we identified a previously uncharacterized lncRNA, named virus inducible lncRNA modulator of interferon response (VILMIR), that was consistently upregulated after in vitro influenza infection across multiple human epithelial cell lines and influenza A virus subtypes. VILMIR was also upregulated after SARS-CoV-2 and RSV infections in vitro. We experimentally confirmed the response of VILMIR to influenza infection and interferon-beta (IFN-β) treatment in the A549 human epithelial cell line and found the expression of VILMIR was robustly induced by IFN-β treatment in a dose and time-specific manner. Single cell RNA-seq analysis of bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients uncovered that VILMIR was upregulated across various cell types including at least five immune cells. The upregulation of VILMIR in immune cells was further confirmed in the human T cell and monocyte cell lines, SUP-T1 and THP-1, after IFN-β treatment. Finally, we found that knockdown of VILMIR expression reduced the magnitude of host transcriptional responses to IFN-β treatment in A549 cells. Together, our results show that VILMIR is a novel interferon-stimulated gene (ISG) that regulates the host interferon response and may be a potential therapeutic target for human respiratory viral infections upon further mechanistic investigation.

overall_design: To investigate the functional role of VILMIR during the human interferon response, VILMIR expression was repressed using CRISPR interference. A549 cells expressing dCas9-KRAB and GFP were transduced with two guide RNAs (gRNAs) targeting the 5’ end of VILMIR (VILMIRg1 and VILMIRg2) along with a negative control gRNA targeting GFP expression (Ctrl). In addition, a STAT1 knockdown cell line (STAT1g1) was included for comparison, as STAT1 is well-known to impact the host IFN response. All cell lines were treated with or without human IFN-β at 1 ng/mL or 10 ng/mL for 6 hours in triplicate. RNA-sequencing and differential expression analysis was then performed to identify the impact of VILMIR knockdown on the host transcriptional response to IFN-β treatment.

first: Kristen

last: John

first: Ian

last: Huntress

first: Ethan

last: Smith

first: Hsuan

last: Chou

first: Tammy

middle: S

last: Tollison

first: Sergio

last: Covarrubias

first: Elisa

last: Crisci

first: Susan

last: Carpenter


title: Acute multi-level response to defective de novo chromatin assembly in S-phase

city: Utrecht

first: Francesca

last: Mattiroli

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.

overall_design: RPE1 degron cell line was treated for 2h with or DMSO (ctrl) or 1µM  dTAG-V1 to induce CHAF1A degradation and disrupt de novo nucleosme assembly. Treated cells where then subjected to Repli-ATAC-seq, VASA-seq and scEdU-seq experiments to assess chromatin accessibility, transcriptome and DNA replication dynamics.

first: Giulia

last: Ricci

first: Vivek

last: Bhardwaj

first: Jeroen

last: van den Berg


title: Serum cytokine and chemokine analysis of patients with treatment-naïve unresectable hepatocellular carcinoma and cancer-free chronic liver disease.

city: Hiroshima-shi

first: Hikaru

last: Nakahara

address: kasumi 1-2-3, minami-ku

institute: Hiroshima University

summary: A total of 116 patients, 96 with treatment-naïve unresectable hepatocellular carcinoma (uHCC) and 20 chronic liver disease without any cancer, were analysed for 17 cytokines and chemokines from serum and analysed for oncological features.

overall_design: In the study presented here, a total of 17 chemokines and cytokines were obtained using a well-defined cohort of 96 uHCC cases and 20 chronic liver disease patients without any cancer as comparison cases , in an attempt to identify prognostic factors.

first: Ryoichi

last: Miura

first: Atsushi

last: Ono


title: Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD [Capture-C]

city: Philadelphia

first: Mitchell

middle: L

last: Conery

state: PA

address: 3615 Civic Center Blvd

department: Center for Spatial and Functional Genomics

institute: Children's Hospital of Philadelphia

summary: To nominate identify tissues relevant to the etiology of bone mineral density we generated ATAC-seq in pediatric hMSC-osteoblasts, hFOB 1.19 cells (hFOBs), osteoclasts, and primary chondrocytes. Leveraging the results of this experiment, we designed a CRISPRi screen in hFOBs with scRNA-seq expression read out. The targets selected for the screen were informed by newly generated Capture-C and bulk RNA-seq from hFOBs.

overall_design: hFOB 1.19 cells were differentiated at 39.5⁰C for 5 days prior to collection for sequencing.

first: James

last: Pippin

first: Matthew

last: Pahl

first: Struan

middle: F

last: Grant


title: Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD [scCRISPR screen]

overall_design: hFOBs constitutitively exprressing dCas9-CRISPRi-KRAB were transfected at a low MOI with a pool of 296 sgRNAs targeted to 89 sites. Cells with sgRNAs were selected for 9 days by puromycin and then differentiated for 5 days at 39.5⁰C prior to sequencing.


title: Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD [ATAC-seq]

overall_design: ATAC-seq was executed in pediatric hMSC-osteoblasts, hFOB 1.19 cells (hFOBs), osteoclasts, and primary chondrocytes in order to study the heritability enrichment of bone mineral density in regulatory regions of a diverse number of cell types. ATAC-seq in hFOBs was also used for identifying regulatory regions to be targeted in a paired CRISPRi screen in hFOBs.


title: Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD [bulk RNA-seq]


title: Single-cell profiling of mouse spleen myeloid cells

city: Maastricht

first: Han

last: Jin

address: P. Debyelaan 25

institute: Maastricht University

summary: Neonates are at higher risk of inflammatory disorders due to their immature immune system. The timely appearance of regulatory immune cells in early life contributes to the control of inflammation in neonates via induction of immunotolerance, the mechanisms underlying which remain poorly understood. In this study, we identified a subset of neonatal monocytes with high expression of transmembrane protein Neuropilin-1 (Nrp1), termed Nrp1high monocytes. Nrp1high monocytes displayed potent immunosuppressive activity and their frequencies declined in age-dependent manner. Deletion of Nrp1 in myeloid cells aggravated the severity of neonatal inflammatory disease necrotizing enterocolitis (NEC). Adoptive transfer of Nrp1high monocytes led to remission of NEC. Mechanistic studies showed that Nrp1, via binding with its ligand semaphorin-4a (Sema4a), induced intracellular p38-MAPK/mTOR signaling and activated transcription factor KLF4. KLF4 activation led to transcription of Nos2 and enhanced the production of nitric oxide, a key mediator of immunosuppression in monocytes. These observations uncover an important immunosuppressive axis in neonatal monocytes, and provides potential therapeutic strategy for inflammatory disorders in neonates.

overall_design: Spleen was harvested from C57BL/6J mice (7 days) and treated with ACK lysis buffer. CD3-CD19-CD11b+ cells were sorted with a MoFlo Astrios EQ Cell Sorter and performed single cell RNA-seq using a Chromium Controller instrument (10× Genomics).

first: Xiaoqing

last: Zheng

first: Jie

last: Zhou


title: ER-stress response in retinal Müller glia occurs significantly earlier than amyloid pathology in in the Alzheimer’s mouse brain and retina

city: Farmington

first: Riqiang

last: Yan

address: 263 Farmington Ave

institute: Uconn Health

summary: Alzheimer’s Disease (AD) pathogenesis is thought to begin up to 20 years before cognitive symptoms appear, suggesting the need for more sensitive diagnostic biomarkers of AD. In this report, we demonstrated pathological changes in retinal Müller glia significantly earlier than amyloid pathology in AD mouse models. By utilizing the knock-in NLGF mouse model, we surprisingly discovered an increase in reticulon 3 (RTN3) protein levels in the NLGF retina as early as postnatal day 30 (P30). Despite RTN3 being a canonically neuronal protein, this increase was noted in the retinal Müller glia, confirmed by immunohistochemical characterization. Further unbiased transcriptomic assays of the P30 NLGF retina revealed that retinal Müller glia were the most sensitive responding cells in this mouse retina, compared to other cell types including photoreceptor cells and ganglion neurons. Pathway analyses of differentially expressed genes in glia cells showed activation of ER stress response via the upregulation of unfolded protein response (UPR) proteins such as ATF4 and CHOP. Early elevation of RTN3 in response to challenges by toxic Aβ likely facilitated UPR. Altogether, these findings suggest that Müller glia act as a sentinel for AD pathology in the retina and should aid for both intervention and diagnosis.

overall_design: Retinas from two wildtype and two nlgf mice were dissected at P30. Cells were isolated into a single-cell suspension and processed using the 10x genomics single-cell RNA sequencing pipeline.

first: Sarah

last: Palko

first: Marc

last: Benoit

first: Royce

last: Mohan


title: RNASeq analysis of CC011, CC074, Trimko, and Trim wt  female mice on 3dpi and 6dpi  after infection with Ebola virus

city: Chapel Hill

first: Alexandra

last: Schaefer

state: NC

address: 3109 MHRC

institute: University of North Carolina at Chapel Hill

summary: Gene expression changes in the liver was studied by RNAseq Results: Strong differences in the liver-specific responses between CC011 and CC074 mice

overall_design: Livers were collected from infected and mock-treated control mice, RNA was isolated and sequenced.

first: Nicholas

last: Catanzaro

first: Cameron

last: Nguyen


title: Auto-sumoylation of the Ubc9 E2 SUMO-conjugating Enzyme Extends Cellular Lifespan

city: Daegu

first: Hong-Yeoul

last: Ryu

state: Buk-Gu

address: Daehak-ro 80

department: Biotechnology

country: South Korea

institute: Kyungpook National University

summary: Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension

overall_design: ChIP of Smt3 in WT and ubc9-RR mutant S.cerevisiae strains

first: Seok-Won

last: Jeoung


title: Single cell RNA sequencing of lineage traced early postnatal myofibroblasts in the adult lung

first: Rachana

last: Radhamani Chandran

state: California

department: Pediatrics

institute: University of California, Los Angeles - Los Angeles, CA

summary: Early postnatal myofibroblasts are a key cell type involved in  postnatal alveolarization. The majority of these  cells undergo apoptosis post-alveolarization. However, some dedifferntaite and  persist in the adult lung. We utilized single cell RNA seq. to understand transcriptomics of these persisted myofibroblasts.

overall_design: Acta2-CreERT2, ROSA26R(ZSgreen/+) mice were injected with 4-OH-TM on P6.5, P8.5 and P10.5, and at P60.5, single cell suspension of the lungs were made. The FACS sorted,  ZSgreen+, cells were subjected to single cell RNA sequencing to assess marker expression in ZSgreen+ cells that were also  SMA+ or SMA

middle: R

last: Chandran

first: Taylor

last: Adams

first: Daniel

last: Greif


title: Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD

first: Mitchell

last: Conery

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

overall_design: Refer to individual Series


title: The pulmonary inflammatory microenvironment at the time of exposure governs innate control of SARS-CoV-2 replication

city: Salvador

first: Artur

middle: Trancoso Lopo

last: Queiroz

address: Rua Waldemar Falcão, 121

country: Brazil

institute: Instituto Gonçalo Moniz

summary: SARS-CoV-2 (SCV2) infection leads to diverse clinical outcomes ranging from asymptomatic to fatal disease. Co-morbidities, age and host genetics together with immune status affect disease severity.  However, the range of innate inflammatory pathways critical for limiting early viral replication and whether the inflammatory history and milieu of the lung microenvironment at the time of exposure impacts early control of SCV2 infection remain largely unexplored. Here, we investigated the roel of prior TLR stimualtion on early viral rpelciation in the lungs of K18 mice

overall_design: Male JAX 34860 (K18-hACE2 Tg) mice, 10-15weeks old, infected with USA-WA1000TCIDd50

first: Paul

middle: J

last: Baker

first: Andrea

last: Bohrer

first: Eduardo

last: Amaral

first: Ehydel

last: Castro

first: Maryonne

last: Snow-Smith

first: Flor

last: Torres-Juárez

last: Rocha

first: Sydnee

last: Gould

first: Cassandra

last: Jordan

first: Jaspal

last: Khillan

first: Kyoungin

last: Cho

first: Reed

last: Johnson

last: Barber

first: Bruno

middle: B

last: Andrade

first: Kerry

last: Hilligan

first: Katrin

last: Mayer-Barber


title: A male-essential microRNA is key for avian sex chromosome dosage compensation

city: Uppsala

first: Amir

last: Fallahshahroudi

address: Gropgränd 4

department: IMBIM

country: Sweden

institute: Uppsala University

summary: Birds have a sex chromosome system in which females are heterogametic (ZW) and males are homogametic (ZZ). The differentiation of avian sex chromosomes from ancestral autosomes entailed the loss of most genes from the W chromosome during evolution. However, to what extent mechanisms evolved that counterbalance the consequences of this extensive gene dosage reduction in female birds has remained unclear. Here we report functional in vivo and evolutionary analyses of a Z-chromosome-linked microRNA (miR-2954) with strongly male-biased expression that was previously proposed to play a key role in sex chromosome dosage compensation1. We knocked out miR-2954 in chicken, which resulted in early embryonic lethality of homozygous knockout males, likely due to the highly specific upregulation of dosage-sensitive Z-linked target genes of miR-2954. Our evolutionary gene expression analyses further revealed that these dosage-sensitive target genes have become upregulated on the single Z in female birds during evolution. Altogether, our work unveils a scenario where evolutionary pressures on females following W gene loss led to the evolution of transcriptional upregulation of dosage-sensitive genes on the Z not only in female but also in male birds. The resulting overabundance of transcripts in males resulting from the combined activity of two dosage-sensitive Z gene copies was in turn offset by the emergence of a highly targeted miR-2954-mediated transcript degradation mechanism during avian evolution. Our findings demonstrate that birds have evolved a unique sex chromosome dosage compensation system in which a microRNA has become essential for male survival.

overall_design: We knocked out a microRNA (miR-2954) in the chickens and studied the developmental phenotypes

first: Mike

last: McGrew

first: Henrick

last: Kaessmann


title: Germinal Center Dark Zone harbors ATR-dependent determinants of T-cell exclusion that are also identified in aggressive lymphoma – Visium_FFPE_Spatial_Trasciptomics_project

city: Palermo

first: Claudio

last: Tripodo

address: Corso Tukory 211

department: PROMISE

institute: Univerisity of Palermo

summary: The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate  expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. How GC polarity is established is poorly understood.  This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features precluding DZ T-cell infiltration. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to estabslish an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.

overall_design:
Whole-genome transcriptomic profiling was performed on formalin-fixed tissue sections of mouse FFPE mesenteric lymph nodes from two samples: one with a knocked Aicda gene (B6.129P2-Aicdatm1(cre)Mnz/J, Jackson, RRID no 007770) and the other under wild-type conditions for Aicda gene (B6N;129-Ilktm1Star/J, Jackson, RRID no 023310).

grant ID : *22145*
grant title: *Linking genetic evolution, stromal and immune contextures to
unravel tumor heterogeneity in aggressive B-cell lymphomas*
name of the funding source:* A.I.R.C. (Associazione Italiana Ricerca
Cancro) *

first: Valeria

last: Cancila

first: Gaia

last: Morello

first: Giorgio

last: Bertolazzi


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [ChIP-seq]

city: Geneva

first: Aurelie

last: Hintermann

address: 30 quai Ernest-Ansermet

department: Genetics and Evolution

country: Switzerland

institute: University of Geneva

summary: The existence of homologies between fins and tetrapod limb skeletal parts as well as of the nature of those mechanisms underlying the transition of the former towards the latter, have been a rich source of discussion for more than a century. While the recent use of gene expression patterns to try and infer evolutionary scenarios has been a popular and successful approach, in particular the distribution of Hox transcript domains, it has failed to provide clearcut evidence as to whether fishes do have bony elements related by ancestry to tetrapod hands and feet. In tetrapods, posterior Hoxd genes transcription in digits is controlled by a well-characterized series of enhancers forming a large regulatory landscape, which has its syntenic counterpart in zebrafish. We show here that the deletion of the orthologous landscape in fishes does not affect the transcription of these genes in fin buds. Instead, it abrogates hoxd expression in the cloaca, an essential structure related to the mammalian uro-genital sinus. We also report that Hoxd gene function in the mammalian uro-genital sinus depends on enhancers located in the same regulatory landscape and thus conclude that an ancestral Hox ‘cloacal’ regulation was co-opted, in tetrapod, as a playground to subsequently accompany the evolution of both external genitals and digits regulatory landscapes, along with the emergence of these developmentally and phylogenetically related structures.

sample_organism:
Danio rerio
Mus musculus

overall_design:
Histone mark ChIP-seq experiments in wild type mouse urogenital sinuses.
Please note that the CTCF ChIP-seq experiments in one mouse sample from GSE194418 and four zebrafish samples from GSE156096 have been re-analyzed:
BioProject accession: PRJNA800459
GEO Sample      SRA Runs        BioSample       Title
==========      ========        =========       =====
GSM5835468   SRR17750150  SAMN25248179  ChIP_CTCF_E105_PT_rep1
BioProject accession: PRJNA656773
GEO Sample      SRA Runs        BioSample       Title
==========      ========        =========       =====
GSM4724551      SRR12435909     SAMN15802273    ChIP-seq in zebrafish wild-type embryos at 24hpf, replicate 1
GSM5344491      SRR14670351     SAMN19371946    ChIP-seq in zebrafish wild-type embryos at 24hpf, replicate 2
GSM5344494      SRR14670354     SAMN19371948    ChIP-seq in zebrafish wild-type embryos at 48hpf, replicate 1
GSM5344495      SRR14670355     SAMN19371947    ChIP-seq in zebrafish wild-type embryos at 48hpf, replicate 2
Please see more details in the 're-analysis_readme.txt'.

first: Aurélie

first: Chase

last: Bolt

first: Lucille

last: Lopez-Delisle

first: Sandra

last: Gitto

first: Bénédicte

last: Mascrez

first: Denis

last: Duboule


title: The role of miRNAs and lncRNAs in regulating transcription in gilthead sea bream (Sparus aurata) myoblasts in response to amino acids and insulin-like growth factor 1 [RNA-seq]

city: Goiania

first: Bruno

middle: Oliveira Silva

last: Duran

state: GO

address: Esperança Avenue, Campus Samambaia, Biological Sciences Institute

department: Department of Histology, Embryology and Cell Biology

institute: Federal University of Goias

summary: In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of genes, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and their regulatory network. AA had a higher impact on gene transcription (1795 genes significantly changed) compared to Igf-1 (385 genes significantly changed). Both treatments stim-ulated the transcription of genes related to muscle differentiation (GO:0042692) and sarcomere components (GO:0030017), but AA stimulated more the DNA replication and cell division (GO:0007049). Notably, four miRNAs (miR-21, miR-146, miR-22b and miR-206) dominated the landscape among 403 expressed miRNAs. Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs) such as miR-133a/b, miR-206, miR-499, miR-1, and miR 27a. Among 111 detected lncRNAs (> 1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting direct regulation; while 30 lncRNAs showed strong correlations and interac-tions with several miRNAs, suggesting their function as miRNA’s sponges. This work is the first step in the identification of ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

overall_design: To obtain the transcriptional profile of gilthead sea bream (Sparus aurata) muscle cells treated with amino acids (AA), IGF1 or cultured in nutrient restriction medium (Control), with n=3 for each experimental group.

first: Isabel

last: García-Pérez

last: Oliveira Silva Duran

first: Maeli

last: Dal-Pai-Silva

first: Daniel

last: Garcia de la Serrana


title: SOCS2 contributes to reduction of the third digit during development of the chicken forelimb

city: Shenyang

first: Xiaoping

last: Li

address: 120 Dongling Rd.

institute: Shenyang Agricultural University

summary: We used the RCAS avian retrovirus to overexpress SOCS2 in the developing chicken limb buds and transcriptome sequencing (mRNA-Seq) revealed changes in expression of genes known to be associated with growth and development in forelimbs with overexpressed SOCS2.

overall_design: mRNA-seq data of injected right wing and uninjected (control) left wing of one chick embryo after SOCS2 overexpression were obtained by Illumina NovaSeq 6000.

first: Shanshan

first: Shibin

last: Bai

first: Zhe

last: Wang


title: A phosphate transporter in VIPergic neurons of the suprachiasmatic nucleus gates locomotor activity during the light/dark transition in mice

city: Zurich

first: Csaba

last: Földy

state: Zurich

address: Winterthurerstrasse 190

department: Brain Research Institute

institute: University of Zurich

summary: The suprachiasmatic nucleus (SCN) encodes time of day through changes in daily firing, with neurons being generally more active during the day and more silent at night. However, molecular mechanisms by which the SCN encodes and times behavior are not fully understood. To identify factors that could encode day/night differences in activity we combined patch-clamp recordings and single-cell sequencing (Patch-RNAseq) of individual SCN neurons in mice.

overall_design: Mice were entrained to a 12:12 light dark cycle (LD 12:12, 12h light, 12h dark) and SCN slices were prepared either at ~ZT0 (for day active neurons) or ~ZT12 (for night active neurons). Multiple recordings and RNA collections were made from the same SCN slice to capture neuronal activity and gene expression. After each recording lasting several minutes, the cytoplasm of the neuron was collected and processed for single-cell RNAseq

first: Sara

last: Pierre-Ferrer

first: Ben

last: Collins

first: David

last: Lukacsovich

first: Shao'Ang

last: Wen

first: Yuchen

last: Cai

first: Jochen

last: Winterer

first: Jun

last: Yan

first: Lene

last: Pedersen

first: Steven

last: Brown


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [RNA-seq]

first: Aurelie

last: Hintermann

overall_design: RNA-seq experiment in E18.5 male and female urogenital sinus from wild type and mutant embryos.

first: Aurélie

first: Chase

last: Bolt

first: Lucille

last: Lopez-Delisle

first: Sandra

last: Gitto

first: Bénédicte

last: Mascrez

first: Denis

last: Duboule


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [CUT&RUN]

overall_design: CUT&RUN experiments against histone marks, in wild type 16hpf zebrafish posterior trunk. Samples were processed according to (10.1038/nprot.2018.015)


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [cHiC]

overall_design: To assay chromatin conformation at the HoxD locus, we conducted capture HiC-seq experiments. We collected urogenital sinuses separately from male and female wild type E18.5 mice. Samples were processed as described in 10.1371/journal.pbio.3000004.


title: EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE WITH THE EMERGENCE OF DIGITS AND GENITALS [HiC-seq re-analysis]

type:
Other
Third-party reanalysis

relation:
SubSeries of: GSE250267
Reanalysis of: GSM4724554
Reanalysis of: GSM4724555
Reanalysis of: GSM5344533
Reanalysis of: GSM5344534
Reanalysis of: GSM4625562

overall_design:
To assay chromatin conformation at the hoxDa locus, we reanalysed published HiC-seq sequencing data conducted on wild type zebrafish collected at either 24 hpf (2 samples from GSE156099 and 1 sample from GSE152741) or 48 hpf (2 samples from GSE156099). All individual samples were aggregated into a single interaction matrix.

BioProject accession: PRJNA656767
GEO Sample SRA Runs  BioSample  Title
========== ========  =========  =====
GSM4724554 SRR12435867  SAMN15802270  HiC in zebrafish wild-type embryos at 48hpf, replicate 1
GSM4724555 SRR12435868  SAMN15802269  HiC in zebrafish wild-type embryos at 48hpf, replicate 2
GSM5344533 SRR14670388  SAMN19372006  HiC in zebrafish wild-type embryos at 24hpf, replicate 1
GSM5344534 SRR14670389  SAMN19372005  HiC in zebrafish wild-type embryos at 24hpf, replicate 2

BioProject accession: PRJNA640313
GEO Sample SRA Runs  BioSample  Title
========== ========  =========  =====
GSM4625562 SRR12044304,SRR12044305,SRR12044306,SRR12044307,SRR12044308,SRR12044309 SAMN15314075  WT_ZF_Embryos_24hpf

Please see more details in the 're-analysis_readme.txt'.


title: Combined deletion of MEN1, ATRX and PTEN triggers pancreatic neuroendocrine tumorigenesis in mice

city: Houston

first: Pawel

last: Mazur

state: Texas

address: 6565 MD Anderson Blvd

institute: MD Anderson Cancer Center

summary: Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of tumors that exhibit an unpredictable and broad spectrum of clinical presentations and biological aggressiveness. Surgical resection is still the only curative therapeutic option for localized PanNET but the majority of patients are diagnosed at an advanced and metastatic stage with limited therapeutic options. Key factors limiting the development of new therapeutics are the extensive heterogeneity of PanNETs and the lack of appropriate clinically relevant models. In that context, genomic sequencing of human PanNETs revealed recurrent mutations and structural alterations in several tumor suppressors.  Here, we demonstrated that combined loss of MEN1, ATRX, and PTEN, tumor suppressors commonly mutated in human PanNETs, triggers pancreatic neuroendocrine tumorigenesis in mice. Histopathological evaluation and gene expression analyses of the developed tumors confirm the presence of PanNET hallmarks and significant overlap in gene expression patterns found in human disease. Thus, we postulate that the presented novel genetically defined neuroendocrine-specific tumor mouse is the first clinically relevant immunocompetent in vivo PanNET model.

overall_design: Gene expression profiles in WT pancreas and MAP mutant mouse PanNET tumors.

first: Xiaoyin

last: Lu

first: Mary

middle: E

last: Fuentes

middle: K


title: Identification of a chromatin-bound ERRα interactome network in mouse liver [RNA-Seq]

city: Montréal

first: Vincent

last: Giguère

state: QC

address: 1160 Pine Ave West, McIntyre Bldg Room 710

department: Goodman Cancer Centre

country: Canada

institute: McGill University

summary:
Objective
Estrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver.
Methods
RIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes
Results
Characterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their co-localization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration.
Conclusions
Our work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.

overall_design: To investigate the cooperative functions of ERRα and HCFC1, Hepa-6 cells were treated with 200nM of siRNAs targeting ERRα (siERRa, si_ERR), HCFC1 (siHCFC1, si_HCF), both ERRα and HCFC1 (siERRa+siHCFC1, si_E_H) or control siRNAs (siC, si_CTL) for 96h. Experiments were performed in triplicate.

first: Charlotte

last: Scholtes

first: Catherine

last: Dufour


title: Identification of a chromatin-bound ERRα interactome network in mouse liver [ChIP-Seq]

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for HCFC1 or IgG control was conducted in Hepa-6 cells treated with 200nM of siRNAs targeting ERRα (siERRa, siE), HCFC1 (siHCFC1, siH) or control siRNAs (siC) for 96h.


title: Identification of a chromatin-bound ERRα interactome network in mouse liver


title: Effect of knockout ERO1α on gene expression in mTORC1 activated mouse embryonic fibroblasts

city: Anhui Province

first: zixi

last: wang

address: Hefei City, Anhui Province

institute: Anhui Medical University

summary: Endoplasmic reticulum oxidoreductase 1 alpha (ERO1α) is an endoplasmic reticulum stress–related gene, which improves cell perseverance against challenges of high levels of protein misfolding during endoplasmic reticulum stress by retaining good activity of oxidative protein folding. Numerous studies have shown abnormal expression of ERo1α in various diseases, but its downstream target are not fully understood. Our work will help in the elucidation of the downstream molecular mechanism of ERO1α.

overall_design: To investigate the downstream effect of ERO1α, we established NTC/T1 null cell lines in which ERO1α has been knocked out by CRISPR-Cas9. We then performed gene expression profiling analysis using data obtained from RNA-seq.

first: Zixi

last: Wang

first: Huaiyuan

last: Zong

first: Xiaojun

last: Zha


title: Rebound after MAPK inhibitor withdrawal in pLGG cells

city: Heidelberg

first: Romain

last: Sigaud

address: Im Neuenheimer Feld

institute: Hopp Children’s Cancer Center Heidelberg (KiTZ)

summary: An in vitro MAPK inhibitor withdrawal rebound model was generated using the patient-derived, PXA-like cell line BT-40 (BRAFV600E, CDKN2Adel). Using this model, we investigated potential cellular intrinsic and extrinsic mechanisms involved in rebound growth in pLGG through multi-omics analysis (RNAseq, phospho-/proteomics).

overall_design: To investigate molecular mechanisms up-/downregulated upon MAPKi treatment and withdrawal, BT-40 cells were treated for 5 days with 5 nM dabrafenib, followed by up to 72h of treatment withdrawal. Five days treatment with DMSO (solvent) serves a control

first: Daniela

last: Kocher

first: Till

last: Milde


title: Coordination of histone chaperones for parental histone segregation and epigenetic inheritance

city: New York

first: zhiguo

last: zhang

state: NY

address: 1130 St. Nicholas Avenue

department: Pediatric and Genetics and Development

institute: Columbia University

summary: Chromatin-based epigenetic memory relies on the accurate distribution of parental histone tetramers to newly replicated DNA strands, serving as templates for chromatin structure duplication. Mcm2, a subunit of the replicative helicase, and the Dpb3/4, subunits of polymerase epsilon, govern parental histone deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here we show in fission yeast that a Mcm2 histone chaperone mutation severely disrupts heterochromatin inheritance, while Dpb3/4 mutations cause only moderate defects. Surprisingly, simultaneous Mcm2 and Dpb3/4 mutations stabilizes heterochromatin inheritance. eSPAN analyses confirm the conservation of Mcm2 and Dpb3/4 functions in parental histone segregation, with their collective absence reducing segregation bias. Furthermore, the FACT histone chaperone also regulates parental histone transfer independently of strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone density, ensuring faithful heterochromatin inheritance. These results underscore the importance of precise parental histone segregation to the lagging strand for epigenetic inheritance and unveil unique properties of parental histone chaperones during DNA replication.

overall_design: Input, BrdU, H3K56ac ChIP-Seq, H3K56ac eSPAN, H3K4me3 ChIP-Seq, H3K4me3 eSPAN, are performed in S.pombe followed by Next Generation Sequencing

first: Yimeng

last: Fang

first: Xu

last: Hua

first: Zhiguo

last: Zhang

first: Songtao

last: Jia


title: Transcriptomic analysis of Sirt3 knock-out mouse embryonic fibroblasts reveals major differences in metabolism and stress-response between sexes

city: Zagreb

first: Robert

last: Beluzic

address: Bijenicka 54

department: Department of Molecular Medicine

country: Croatia

institute: Rudjer Boskovic Institute

summary: In this study, we investigated sex-specific differences in gene expression caused by Sirt3 knockout. SIRT3, a mitochondrial deacetylase, plays a crucial role in maintaining cellular health. Through activation or suppression of a large number of target proteins, it integrates various metabolic processes, including energy production and antioxidant defense. Using NGS mRNA-seq on wild-type and Sirt3-knock-out male and female mouse embryonic fibroblasts (MEFs) we detected significant changes in both global gene signature and affected cellular pathways. More importantly, we describe major differences in metabolic status of male and female KO MEFs which include fundamental processes like glycolysis, TCA cycle and beta-oxidation. We describe a sex-specific response to Sirt3 loss including maintainance of cellular oxidative status, Hif1a stabilization  and induction of Integrated Stress Response (ISR). While female MEFs are significantly more able to tolerate Sirt3 loss, male cells adapt to knock-out by entering a persistent state of cellular stress. Considering many known and potential roles of Sirt3 in metabolism of both normal and cancer cells, aging and longevity, this study emphasizes the crucial role of including sex as a variable in biomedical research.

overall_design: Sirt3 knock-out mouse embryonic fibroblasts were isolated from 13 days old embryos of 129S1/SvImJ WT (Stock No: 002448 Jackson Laboratory, Bar Harbor, ME, USA)  Sirt3+/+ and Sirt3-/- (Stock No: 012755, Jackson Laboratory, Bar Harbor, ME, USA) female mice and stable cells were generated using SV40 promoter-containing plasmid. To investigate sex-specific effects of Sirt3 knock-out, we performed mRNA-seq, in triplicates, on wild-type male (WT_M), wild-type female (WT_F), Sirt3 knock-out male (KO_M) and female (KO_F) cells.

first: Iva

last: Skrinjar

first: Marija

last: Pinteric

first: Marijana

last: Hadzija

first: Ena

last: Simunic

first: Tihomir

last: Balog

first: Sandra

last: Sobocanec


title: Influences of chronic hypoxia and hypoxic-inducible factor 2A on mouse myoblasts

city: Athens

first: Hang

last: Yin

state: GA

address: 325 Riverbend Rd

department: Center for Molecular Medicine

institute: The University of Georgia

summary: The extracellular oxygen levels play a crucial role in the control of cellular metabolism and behaviors under various physiological and pathological conditions. When mammalian cells sense insufficient oxygen levels, one of the most important responses is to stabilize hypoxic-inducible factors. While the functions of HIF-1A and acute hypoxia signaling have been extensively studied in many cell types, the impacts of HIF-2A and chronic hypoxia on skeletal muscle myoblasts have not been fully understood.

overall_design: This study provides the first sketch of the above by comparing global gene expression values (1) in mouse myoblasts (derived from FACS-sorted muscle satellite cells) adapted to “normoxia” (20% pO2), “mild hypoxia” (4% pO2), and “severe hypoxia” (1% pO2) cultured conditions for >2 weeks; and 2) in mouse myoblasts treated with HIF-2A inhibitor PT2385 (1 uM) or a vehicle solution (0.1% DMSO) under each above conditions.


title: Accumulation of Notch3 extracellular domain and not aberrant NOTCH3 signaling is the key driver of CADASIL arterial pathology

city: Paris

first: Anne

last: JOUTEL

address: 102 rue de la Sante

institute: Inserm

summary: Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 (Notch3ECD) receptor are the two core features of CADASIL, the most common genetic cerebral small vessel disease. Although CADASIL is known to be caused by highly stereotyped dominant mutations in NOTCH3, the relationship between NOTCH3 receptor activity, Notch3ECD  accumulation and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using novel histopathological and multiscale imaging modalities, we quantified previously undetectable CADASIL-driven focal arterial SMC loss in the central nervous system of mice expressing the archetypal Arg169Cys CADASIL mutation. Notably, we found more severe arterial pathology and greater Notch3ECD accumulation in transgenic model mice overexpressing the mutation on a homozygous wild-type Notch3 background (TgNotch3R169C) than in knock-in model Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. We further showed that wild-type Notch3ECD co-aggregated with mutant Notch3ECD and that elimination of one copy of wild-type Notch3 in TgNotch3R169C mice was sufficient to attenuate Notch3ECD accumulation and arterial pathology. Importantly, RNA sequencing revealed no substantial change in the expression of Notch3-regulated genes in TgNotch3R169C brain arteries. Collectively, our results provide compelling evidence that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, and not aberrant NOTCH3 signaling, is the key driver of arterial SMC loss in CADASIL, thus identifying NOTCH3-lowering approaches as candidate therapeutic strategies.

overall_design: To disentangle whether a loss or a gain of NOTCH3 activity or a neomorphic effect are involved in CADASIL-associated arterial SMC loss, we profiled the transcriptome of brain arteries dissected from TgNotch3R169C aged 12 months, Notch3 deficient (Notch3-/-) aged 3 months and their WT respective age-matched littermates using bulk RNA-sequencing. We then performed comparative gene expression profiling of RNAseq data.

first: Nicolas

last: Dupré

first: Florian

last: Gueniot

first: Valérie

last: Domenga-Denier

first: Christelle

last: Nilles

first: Virginie

last: Dubosclard

last: Morgenthaler-Roth

first: Céline

last: Keime

first: Lydia

last: Danglot

last: Joutel


title: ATACseq for Young and Old mouse liver

city: La Jolla

first: Xue

last: Lei

address: 10901 N Torrey Pines Rd

institute: SBP medical discovery institute

summary: Our goal was to identify how aging affects chromatin accessability in the mouse liver.

overall_design: C57BL/6 mice were euthanized using CO2 and cervical dislocation.  Whole liver was excised and maintained on ice cold DMEM and nuclei were isolated while tissue was fresh. Tagmentation was conducted for 30 minutes at 37 degrees Celsius followed by DNA isolation and library preperation.

first: Aaron

last: Havas

first: Peter

last: Adams

first: Neil

last: Robertson

first: Brandon

last: Sos

first: Kun

last: Zhang


title: An integrated toolkit for human microglia functional genomics

first: Falak

last: Sher

state: New York

address: 630 W 168th street

summary:
Microglia, the brain’s resident immune cells, play vital roles in brain development, and disorders like Alzheimer’s disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9.
In this study will generated iMG from human iPSCs using our newly developed protocol. We used single cell RNA-Seq to study the transcriptomic profiles of our newly formed iMG. The single cell RNA-Seq data under this accession is from wild type iMG (Hashtag 7) and OTHER KO iMG (Hashtag 8)

overall_design: iPSC-derived microglia (iMG) were harvested from in vitro cultures, washed with PBS and hashtaged  with hash tagging antibodies as: WT: Hashtag 7, OTHER KO Hashtag 8. scRNA-Seq was performed using 10X Genomics Chromium Next GEM single-cell 3' Reagent kit v3.1 leveraging its Feature Barcode technology.

first: Imdadul

last: Haq

first: Jason

middle: C

last: Ngo

first: Vilas

last: Menon


title: The role of miRNAs and lncRNAs in regulating transcription in gilthead sea bream (Sparus aurata) myoblasts in response to amino acids and insulin-like growth factor 1 [miRNA-seq]

first: Bruno

last: Duran

overall_design: To obtain the microRNAome profile of gilthead sea bream (Sparus aurata) muscle cells treated with amino acids (AA), IGF1 or cultured in nutrient restriction medium (Control), with n=3 for each experimental group.

first: Isabel

last: García-Pérez

last: Oliveira Silva Duran

first: Maeli

last: Dal-Pai-Silva

first: Daniel

last: Garcia de la Serrana


title: SMARCA4 loss and mutated β-catenin induce proliferative lesions in the murine embryonic cerebellum [scRNA-seq]

city: Hamburg

first: Ulrich

last: Schüller

address: Martinistraße 52

institute: University Medical Center Hamburg-Eppendorf

summary: Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in around 20% of WNT MB, but their functional role is mostly unknown. We therefore amended previously described Blbp cre::Ctnnb1(ex3)fl/wt mice by the introduction of a floxed Smarca4 allele. Unexpectedly, mutated β-catenin on its own induced severe developmental phenotypes in Blbp cre::Ctnnb1(ex3)fl/wt mice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and non-proliferative cell accumulations in the brain stem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. Respective Blbp cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants developed large proliferative lesions in the cerebellum evolving from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation profiling and single-cell RNA sequencing suggested an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulated WNT signaling, altered actin/cytoskeleton organization, and reduced neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult recipient mice. However, in cerebellar explant cultures, mutant cells displayed significantly increased proliferation, suggesting an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step towards the unravelling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency.

overall_design: Whole cerebella of E16.5 embryos (cre-negative controls and Blbp‑cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants with cerebellar lesions) were analyzed by single-cell RNA sequencing

first: Carolin

last: Göbel

first: Melanie

last: Schoof

first: Dörthe

last: Holdhof

first: Michael

last: Spohn


title: The role of miRNAs and lncRNAs in regulating transcription in gilthead sea bream (Sparus aurata) myoblasts in response to amino acids and insulin-like growth factor 1

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

overall_design: Refer to individual Series


title: SMARCA4 loss and mutated β-catenin induce proliferative lesions in the murine embryonic cerebellum

summary: Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) subgroup are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in around 20 % of WNT MB but their functional role is mostly unknown. We therefore amended previously described Blbp-cre::Ctnnb1(ex3)fl/wt mice by the introduction of a floxed Smarca4 allele. In contrast to existing literature, even mutated β-catenin on its own in our Blbp-cre::Ctnnb1(ex3)fl/wt mice induced severe developmental phenotypes including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and non-proliferative cell accumulations in brain stem and cerebellum. An additional homozygous loss of SMARCA4 even resulted in prenatal death for most mice. Interestingly, Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants developed big proliferative lesions in the cerebellum that evolved from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation analysis and single-cell RNA sequencing suggest an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulation of WNT signaling and altered actin/cytoskeleton organization and neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult recipient mice. However, mutant cells displayed increased proliferation compared to controls in cerebellar explant culture, indicating an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step in unravelling tumorigenic mechanisms induced by aberrant WNT signaling and a SMARCA4 deficiency.

overall_design: We performed global DNA methylation analysis of E16.5 embryos (Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants and cre-negative controls) to unravel epigenetically deregulated pathways in the histologically evident cerebellar lesions of mutants.


title: p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response [ChIP-seq]

city: Badalona

first: Biola

middle: M

last: Javierre

state: Barcelona

address: Ctra de Can Ruti, Camí de les Escoles , s/n

country: Spain

institute: Josep Carreras Leukaemia Research Institute

summary: Activation of the p53 tumor suppressor triggers a transcriptional program to control cellular response to stress. However, the molecular mechanisms by which p53 controls gene transcription are not completely understood. Here, using a multi-omics integration framework, we uncover the critical role of spatio-temporal genome architecture in this process. We demonstrate that p53 drives direct and indirect changes in genome compartments, topologically associating domains and DNA loops within minutes of its activation, which escort the p53 transcriptional program along time. Focused on p53-bound enhancers, we report a core transcriptional program of 340 genes directly regulated by p53 over distance, most of these not previously identified. Finally, we showcase that p53 controls transcription of distal genes through newly formed and pre-existing enhancer-promoter loops in a cohesin dependent manner. Taken together, our findings demonstrate a previously unappreciated architectural role of p53 as regulator at distinct topological layers and provide a reliable set of new p53 direct target genes that may help future designs of p53-based cancer therapies.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27ac and H3K4me1 in HCT116 cells.

first: François

last: Serra

first: Andrea

last: Nieto-Aliseda

first: Llorenç

last: Rovirosa

first: Mónica

last: Cabrera-Pasadas

first: Lucía

last: Fanlo

first: Aleksey

last: Lazarenkov

first: Blanca

last: Urmeneta

first: Andrei

last: Okorokov

first: Peter

last: Fraser

first: Jose

last: Sardina

first: Alfonso

last: Valencia


title: RNAseq for Young Old mouse liver hepatocytes with and without high fat diet

first: Xue

last: Lei

summary: Our Goal was to determine how hepatocytes of young and old mice differentially respond to 9-weeks of high fat diet vs normal chow.

overall_design: Upon receipt mice were rested for at least 2-weeks prior to initiation of experiment in which mice were maintained on either High fat diet (58y1, TestDiet) or  normal CHOW (Teklad global 18% protein) for 9-weeks prior to euthanasia and hepatocyte isolation.

last: Adams

first: Aaron

last: Havas


title: Critical Role of Ferritin-Mediated Iron Homeostasis in the Maintenance of Hematopoietic Stem Cells and Leukemia Stem Cells

city: Guangzhou

first: Qiang

last: Zhan

address: 601 W.Huangpu Avenue

institute: Jinan University

summary: Iron metabolism has emerged as a critical factor in cell viability, both in normal and pathological contexts. However, the intricate relationship between iron metabolism and the maintenance of adult stem cells and cancer stem cells remains incompletely understood. The ferritin complex, responsible for intracellular iron storage and buffering, plays a pivotal role in this process. Our research provides insights into the regulatory role of ferritin-mediated iron homeostasis in hematopoietic stem cells (HSCs).

overall_design: We generate the conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system to determine the function of iron in HSCs. Fth1-deleted HSC and WT HSC were isolated from 2-month-old Fth1 deletion and WT mice (3-4 repetitions per set), and the bulk RNA-seq were performed. Index of the reference genome was built using Hisat2 v2.0.4 and paired-end clean reads were aligned to the reference genome GRCm38 using Hisat2 v2.0.4. HTSeq v0.9.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.


title: p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response [RNA-seq]


title: VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis

city: Nashville

first: Jeff

last: Rathmell

state: TN

address: 1211 Medical Center Drive

department: PMI

institute: Vanderbilt University Medical Center

summary: Clear cell renal cell carcinoma (ccRCC) is an aggressive disease characterized by dysregulated hypoxia signaling, metabolic defects, and a complex tumor microenvironment (TME) highly enriched in lymphoid and myeloid immune cells. Loss of the oxygen sensing gene, von Hippel Lindau (VHL), is a critical early event in ccRCC pathogenesis and promotes stabilization of hypoxia inducible factors (HIF) that upregulate pro-growth signaling pathways, including angiogenesis and aerobic glycolysis. However, whether VHL loss in cancer cells impacts the composition, metabolism, or function of immune cells in the TME remains unclear. Using Vhl wildtype (WT) and knockout (KO) in vivo murine kidney cancer models, we found that Vhl KO tumors were less proliferative and more infiltrated by immune cells. Tumor-associated macrophages (TAM) demonstrated enhanced proinflammatory transcriptional signatures from Vhl deficient tumors, and intratumoral myeloid cells residing in the Vhl KO TME showed increased in vivo glucose consumption. Vhl loss did not confer increased in vivo glucose uptake in cancer cells or lymphocytes, or detectable changes in metabolites in the interstitial fluid. Mechanistically, enhanced secretion of the chemokine, CX3CL1, was observed in Vhl KO cancer cells and its cognate receptor, CX3CR1, was significantly elevated on myeloid cells residing in the TME of Vhl deficient tumors. Human ccRCC tumors exhibit high expression of CX3CL1 and CX3CR1 as well as enhanced myeloid metabolism and proinflammatory properties. Here, we identify the importance of cancer cell-specific genetic features to drive environmental reprograming and shape the tumor immune landscape and identify a potential therapeutic target in the treatment of ccRCC.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Samples include primary subcutaneous tumor specimens from either VHL WT or VHL KO genetically distinct tumors.


title: p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response [HiC]

last: Javierre


title: Characterization of immortalized human lacrimal gland epithelial cells in 2D and 3D cell culture models

city: Brno

first: Core Facility

last: Bioinformatics

address: Kamenice 5

country: Czech Republic

institute: CEITEC - Masaryk University

summary:
In humans, the lacrimal gland produces the aqueous component of the tear film, which e.g. moistens and nourishes the ocular surface to maintain ophthalmic health.
Decreased production of the aqueous component leads to the development of dry eye disease, which affects over 250 million people worldwide.
Despite the impact on patients, the availability of primary human material to study underlying disease mechanisms is severely constrained.
Here, we report the development of an immortalized human lacrimal gland epithelial cell line that improves accessibility to study the molecular pathogenesis-mechanisms of dry eye disease and link them to causal treatments.

overall_design: Comparative gene expression analysis of three SV40-positive cell clones in four replicates compared to human lacrimal gland tissue from four donors (clone 1, clone 3, clone 6 and control).

first: Gleixner

last: Sophie

first: Arnold

last: Philipp

first: Blavet

last: Nicolas


title: Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

city: Montreal

first: Jacquetta

last: Trasler

state: Quebec

address: 1001 Decarie Boul, Block E, ES1.4380

department: Pharmacology & Therapeutics; Human Genetics

type: Methylation profiling by high throughput sequencing

summary: 5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T), results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to: 1) characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2mg folic acid/kg diet) and 2) assess the effects of folic acid deficiency (0.3mg/kg diet) and supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing, Illumina Mouse Methylation Array and whole genome bisulfite sequencing. Reproductive parameters and imprinted gene methylation were unaffected by genotype or diets. The largest effect was due to genotype, with sperm from 677TT mice showing more hypo- than hypermethylation. Folate-deficient diets resulted in sperm hyper- and hypomethylation in CC and TT mice. Folic acid supplementation caused mostly hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

overall_design: The experimental design consists of six groups of male C57Bl/6J mice: Mthfr TT mice and wild-type mice as control (Mthfr CC) on 3 different diets. The diets consisted of control (CD, 2mg folic acid/kg diet), folate-deficient (FD, 0.3mg folic acid/kg diet) and moderate dose folic acid-supplemented (FASD, 5-fold supplemented: 10 mg folic acid/kg diet) diets. Mice were placed on their respective diets starting at four weeks of age and maintained on them for four months.  At the end of diet treatments mice were sacrificed and tissues and organs were collected.

first: Edgar

last: Martínez Duncker Rebolledo

first: Donovan

last: Chan

first: Karen

last: Christensen

first: Alaina

last: Reagan

first: Gareth

last: Howell

first: Rima

last: Rozen


title: First exploration of the altered microbial gut-lung axis in the pathogenesis of human refractory chronic cough

city: Firenze

first: Matteo

last: Ramazzotti

address: viale Morgagni 50

department: Experimental and Clinical Biomedical Sciences

institute: University of Florence

summary: v3-v4 16S rRNA sequencing was used to characterize both gut and oral microbiota composition of RCC (refractory chronic cough) patients and matched healthy controls (HC). The groups are matched in age and gender.

overall_design: Total DNA was extracted from stool and saliva of 10 patients and 10 healthy subjects, amplified to collect amplicons of variable V3–V4 regions (primers 341F and 805R) of the bacterial 16s rRNA gene and sequenced with MiSeq (2x300bp) Illumina platform.

first: Amedeo

last: Amedei

first: Simone

last: Baldi

first: Leandro

last: Di Gloria


title: Robust Host Engineering: Enhancing Escherichia coli´s Abiotic Stress Resistance through Ornithine Lipid Membrane Modification

city: Cuernavaca

first: Jose

last: Utrilla

state: Morelos

address: Av. Universidad sn

department: Centro de Ciencias Genómicas

country: Mexico

institute: Universidad Nacional Autonoma de México

summary: Transcriptome analysis revealed that strains had differentially expressed stress and membrane-related genes compared to the control strain

overall_design: mRNA of exponentially growing (11h) E. coli mutants in MOPS pH 7.4  and M9 pH 5.4 minimal medium supplemented with glucose

first: Leidy

middle: P

last: Bedoya Pérez

first: Omar

last: Aguilar Vera

first: José

last: Utrilla Carreri

first: Christian

last: Sohlenkamp


title: The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality

city: Moscow

first: Natalia

last: Klimenko

address: 34/5 Vavilova Street

country: Russia

institute: Institute of Gene Biology (IGB) of the Russian Academy of Sciences

summary: CTCF is the main architectural protein found in the majority of examined bilaterians. CTCFs of various organisms contain unstructured N-terminal dimerization domain (DD) along with clusters consisting of eleven zinc-finger domains of C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCF share sequence homology only in five C2H2 domains that specifically bind to conserved 15 bp motif. By using CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene, we generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected solely by the deletion of the N-terminal DD. Also, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression level compared to the dCTCF variants, it efficiently bound to chromatin similar to the wild type (wt) protein. Our results suggest that one of the evolutionarily conserved function of the unstructured dimerization domain is to recruit dCTCF to its binding sites in vivo.

overall_design:
ChIP-seq signal of the dCTCF and CP190 proteins occupancy in dCTCFwt-HA, dCTCFΔ80-170-HA, dCTCFΔ132-170-HA and dCTCFhN lines.
Please note that processed data file generated from both replicates is linked to the corresponding *_1 sample records.

first: O

last: Maksimenko

first: N