title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

city: Toronto

first: Maria

middle: Cristina

last: Nostro

address: 101 College Street

institute: McEwen Stem Cell Institute

summary: To improve β-like cell differentiation from human pluripotent stem cells (hPSCs), we aimed to substitute a mechanistically elusive compound – nicotinamide (NA), which promotes pancreatic progenitor (PP) commitment, with a pathway-specific compound. By performing a low-throughput chemical screening, we demonstrated that tankyrase inhibitors (TNKSi) can replace NA and efficiently generate PPs. Surprisingly, highly selective TNKSi, such as WIKI4, generate PPs that give rise to islet-like populations with improved β-like cell frequencies and glucose responsiveness compared to NA-derived PPs. Characterization of differentially expressed genes (DEG) between WIKI4 and NA treatment revealed that WIKI4-derived PPs expressed higher levels of genes associated with integrin signalling and actin cytoskeleton pathways. Our findings show that compared to NA, WIKI4 treatment promotes changes in cell shape and motility. Together, our results provide novel insights into pancreatic differentiation and a model that better recapitulates native events associated with pancreas and endocrine commitment.

overall_design: H1 Human pluripotent stem cells were differentiated to stage 3 PDX1 + endoderm according to our published protocol (Nostro et al, Stem Cell Reports 2015) and differentiated to stage 4 Pancreatic Progenitors using a combination of Noggin, EGF and either Nicotinamide (NA) or WIKI4 (WIKI). Day 13 pancreatic progenitors were collected for bulk RNA sequencing

first: Frankie

last: Poon

first: Rangarajan

last: Sambathkumar

first: Roman

last: Korytnikov

first: Yasaman

last: Aghazadeh

first: Amanda

last: Oakie

first: Paraish

last: Misra

first: Farida

last: Sarangi

first: M. Cristina


title: DNA immunoprecipitation sequencing (DIP-seq) of Xenopus tropicalis blastula embryos using 5mC, 5hmC, 5fC and 5caC antibodies

status: Public on Aug 29 2024

city: Mainz

first: Emil

last: Karaulanov

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology

type: Methylation profiling by high throughput sequencing

summary: The oxC-marks 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) are iterative oxidation products of 5-methylcytosine (5mC) which might also function as epigenetic marks on their own to regulate gene expression. To characterize the genomic profiles of oxC-marks in Xenopus tropicalis blastula embryos, we carried out DIP-seq using 5mC, 5hmC, 5fC and 5caC antibodies.

overall_design: Genomic DNA from stage 9 Xenopus tropicalis embryos was analyzed by DIP-seq in triplicates using 5mC, 5hmC, 5fC and 5caC antibodies. Input genomic DNA and IgG-immunoprecipitated samples served as specificity controls.


title: Hippocampal CA1 Pyramidal Neurons Display Sublayer and Circuitry Dependent Degenerative Expression Profiles in Aged Female Down Syndrome Mice

status: Public on Aug 26 2024

city: Orangeburg

first: Stephen

middle: D.

last: Ginsberg

state: New York

address: 140 Old Orangeburg Road

department: Centre for Dementia Research

country: USA

institute: Research Foundation for Mental Hygiene

type: Expression profiling by high throughput sequencing

summary: People with Down syndrome (DS) have intellectual disability (ID) and develop hallmark Alzheimer’s disease (AD) pathology during midlife. There are several circuits underlying memory and executive function in the DS and AD brain that are particularly vulnerable and degenerate early in disease, most notably the septohippocampal circuit and the trisynaptic loop in the hippocampus. A fundamental lack of knowledge exists as to the etiology and mechanisms of disease progression within these critical circuits vulnerable to degeneration in DS, AD, and relevant models. This is compounded by new evidence that suggests spatial localization of neurons has profound effects on activity and innervation within the hippocampal CA1 region. We postulated gene expression changes in a DS mouse model, at a time that these circuits of input to the CA1 are degraded, would have a significant effect on gene expression in CA1 pyramidal neurons. Further, this dysfunction may be specific to spatial localization and innervation. Laser capture microscopy on pyramidal neurons from CA1 was performed, isolating the entire CA1 pyramidal neuron layer, which was compared to select populations of deep and superficial pyramidal neurons from CA1a (distal CA1, adjacent to the subiculum). RNA-seq and bioinformatic analysis was performed where profound differences in dysregulation in the DS mouse model based on their spatial location and postulated circuitry were examined.

overall_design:
To determine if circuitry will effect gene expression in a mouse model of DS during degeneration, the CA1 pyramidal neuron region was analyzed in 2N (disomic control; N=11 in 2 batches) and Ts2 (N=11 in 2 batches) Female mice at 11 MO.  LCM was performed on the excitatory pyramidal neuron layer from the entire rostral CA1 region  and compared to the distal end of the rostral CA1 deep layer pyramidal neurons and the distal end of the rostral CA1 superficial layer pyramdial neurons.  RNA was purified from the isolated neurons and RNA-seq library preparation was performed, followed by RNA-seq and bioinformatic analysis.
We compared the effect of genotype on gene expression within each region of CA1 neurons collected by bioinformatic analysis to determine convergent and divergent gene expression in each region to examine changes in hippocampal CA1 gene expression in the aged DS mouse model. Circuitry effects due to innervation were taken into account for analysis of divergent gene expression between deep and superficial neurons compared to the CA1 excitatory neurons as a whole in the Ts2 mice compared to their 2N littermates.

first: Melissa

middle: J

last: Alldred

first: Harshitha

last: Pidikiti

first: Kyrillos

last: Ibrahim

first: Sanghan

last: Lee

first: Adriana

last: Heguy

first: Gabriel

last: Hoffmann

first: Grace

middle: E

last: Stutzmann

middle: D


title: Renin Regulatory Pathway Inhibition Epigenetically Silences *Ren1* (scRNA-Seq)

city: Charlottesville

first: Jason

middle: Paul

last: Smith

state: VA

address: 409 Lane Rd

department: Pediatrics

institute: University of Virginia

summary: Background: Renin-expressing cells are myoendocrine cells crucial for survival which detect changes in blood pressure and release renin to maintain homeostasis. Renin is regulated through several pathways including cAMP, p300/CBP, and BET proteins including Brd4. Binding to the cAMP-responsive element in the renin enhancer region amplifies renin transcription and relies on each of these factors for an appropriate response. The specific regulatory changes that occur following inhibition of these pathways remains understudied. Therefore, we aimed to evaluate chromatin changes and gene signatures that occur following inhibition of renin regulatory factors. Methods: We treated As4.1 cells (a tumoral cell line that constitutively expresses renin) with three inhibitors (H89: PKA inhibition; JQ1: Brd4 inhibition; A-485: p300/CBP inhibition) that target required factors for renin transcriptional regulation. We then performed ATAC-seq, scRNA-seq, and ChIP-seq for H3K27ac and P300 binding on biological replicates of treated and control As4.1 cells. Results: Ren1 expression is significantly, but transiently, reduced following each inhibitory treatment. Further, PKA inhibition leads to corresponding losses in H3K27ac and p300 binding at the locus. A restricted set of nine genes with overlapping dynamically accessible regions, differential gene expression, and H3K27ac and p300 binding were identified with roles across three primary renin regulatory paradigms.   Conclusions: The data shows that renin expression is regulated through a switch from an active to poised state of epigenetic control, a shift toward a less differentiated cellular identity via the loss of enhancer elements, and the disruption of cAMP, baroreceptor, and Notch mediated renin regulatory pathways.

overall_design: The regulation of renin was evaluated in As4.1 cells, a mouse cell line which expresses high levels of renin mRNA, by treating with either a control compound (DMSO) or separate inhibitors to either PKA, Brd4, or p300/CBP.  The regulatory effects were evaluated using ATAC-seq, scRNA-seq, and ChIP-seq for H3K27ac and P300 binding.

middle: P

first: Robert

last: Paxton

first: Silvia

last: Medrano

first: Nathan

middle: C

last: Sheffield

first: Maria

middle: S

last: Sequeira-Lopez

first: R

middle: A

last: Gomez


title: Renin Regulatory Pathway Inhibition Epigenetically Silences *Ren1* (CUTnTag)

type: Genome binding/occupancy profiling by high throughput sequencing


title: Renin Regulatory Pathway Inhibition Epigenetically Silences *Ren1* (ATAC-Seq)


title: ATRX reduces the accessibility of HCMV genomes during lytic infections

status: Public on Aug 28 2024

city: Madison

last: Kalejta

state: Wisconsin

address: 1525 Linden Dr

department: IMV

institute: University of Wisconsin-Madison

summary: We investigated the role of the chromatin remodeling protein ATRX on chromatin accessibility of HCMV genomes during the IE phase of lytic infections

overall_design: ATAC-seq was performed on shScr and shATRX fibroblasts infected with AD169 for 3 hours

middle: M

last: Walter

middle: F


title: Renin Regulatory Pathway Inhibition Epigenetically Silences *Ren1* (ChIP-Seq)


title: Transcriptomic profiling in NAT10 knockdown acute myeloid leukemia cells

city: Guangzhou

first: Huilin

last: Huang

address: #651 East Dong-feng Road

country: China

institute: Sun Yat-Sen University Cancer Center

summary: RNA modification represents an important post-transcriptional regulatory mechanism in acute myeloid leukemia (AML), however the function and mechanism of RNA acetylation ac4C in AML remains elusive. Here, we report that NAT10, as the ac4C writing enzyme, plays a critical oncogenic function in AML and represents a promising therapeutic target for AML. To determine the impact of NAT10-mediated ac4C on gene expression, we conducted high throughput RNA-sequencing (RNA-seq) in NAT10-knockdown and control MOLM13 cells.

overall_design: To investigate the impact of NAT10 on gene expression in MOLM13 cells, we establised NAT10-knockdown MOLM13 cell lines by two shRNAs (NAT10-sh1 and NAT10-sh2) together with control MOLM13 cells (shNS). We then performed RNA sequencing (RNA-seq) in NAT10-knockdown and control MOLM13 cells.

first: Hengyou

last: Weng

first: Subo

last: Zhang

first: Yushuai

last: Wang


title: NAT10-mediated mRNA N4-acetylcitidine (ac4C) Promotes Stemness and Leukemogenesis in Acute Myeloid Leukemia

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

overall_design: Refer to individual Series


title: Transcriptomic ac4C profiling in NAT10 knockdown acute myeloid leukemia cells

summary: RNA modification represents an important post-transcriptional regulatory mechanism in acute myeloid leukemia (AML), however the function and mechanism of RNA acetylation ac4C in AML remains elusive. Here, we report that NAT10, as the ac4C writing enzyme, plays a critical oncogenic function in AML and represents a promising therapeutic target for AML. To understand the mechanisms underlying the function of NAT10 as an RNA ac4C writer in AML, we profiled ac4C modification in the transcriptome of MOLM13 cells using a refined ac4C RNA immunoprecipitation and high throughput sequencing (RacRIP-seq) protocol, and performed systematic calibration with a modification-free control library  generated from the in vitro- transcribed MOLM13 transcriptome (referred to as IVT control) to eliminate most of the false- positive signals. We also applied the RacRIP-seq in NAT10 knockdown and control MOLM13 cells to characterize NAT10 targets.

overall_design: We performed a refined ac4C RNA immunoprecipitation and high throughput sequencing (RacRIP-seq) in MOLM13 cells and systematic calibration with a modification-free control library  generated from the in vitro- transcribed MOLM13 transcriptome (referred to as IVT control). We also applied the RacRIP-seq in NAT10 knockdown (shNAT10-1 and shNAT10-2) and control (shNS) MOLM13 cells to characterize NAT10 targets.


title: Patient iPSC models reveal glia-intrinsic phenotypes in multiple sclerosis

city: Cleveland

first: Benjamin

last: Clayton

state: OH

address: 2109 Adelbert Rd BRB739A

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS), resulting in neurological disability that worsens over time. While progress has been made in defining the immune system’s role in MS pathophysiology, the contribution of intrinsic CNS-cell dysfunction remains unclear. Here, we generated a collection of iPSC lines from people with MS spanning diverse clinical subtypes and differentiated them into glia-enriched cultures. Using single-cell transcriptomic profiling and orthogonal analyses, we observed several distinguishing characteristics of MS cultures pointing to glia-intrinsic disease mechanisms. We found that primary progressive MS-derived cultures contained fewer oligodendrocytes. Moreover, MS-derived oligodendrocyte lineage cells and astrocytes showed increased expression of immune and inflammatory genes, matching those of glia from MS postmortem brains. Thus, iPSC-derived MS models provide a unique platform for dissecting glial contributions to disease phenotypes independent of the peripheral immune system and identify potential glia-specific targets for therapeutic intervention.

overall_design: Human iPSCs from healthy controls and people with MS were differentiated into glia enriched cultures (Douvaras and Fossati, Nat. Protocol 2015 PMID:26134954) and then analyzed by single-cell RNAseq using 10x Genomics.

middle: L

first: Lilianne

last: Barbar

first: Paul

last: Tesar

first: Valentina

last: Fossati


title: Pol III transcribed genes, RNA-Seq, male mouse, duodenum

city: London

first: Nazif

last: Alic

address: Gower St

country: United Kingdom

institute: UCL

type: Non-coding RNA profiling by high throughput sequencing

summary: RNA-Seq of small (Pol III-transcribed) RNAs in duodenum of male mice, Pol III heterozygous mutant versus wild-type

overall_design: RNAs isolated, size selected and demethylated before samples sent to Glasgow Polyomics. RNAseq

first: Yasir

last: Malik

first: Yavuz

last: Kulaberoglu

first: Gillian

last: Borland

first: Colin

last: Selman

first: Jennifer

last: Tullet


title: RNA polymerase III in ageing, in C.elegans, fruit flies and mice

sample_organism:
Caenorhabditis elegans
Drosophila melanogaster
Mus musculus


title: Pol III transcribed genes, RNA-Seq, female mouse, duodenum

summary: RNA-Seq of small (Pol III-transcribed) RNAs in duodenum of female mice, Pol III heterozygous mutant versus wild-type


title: Gene expression profile using Rhapsody/scRNA-seq of brain metastasis-associated immune cells.

city: Derio

first: Paloma

last: Velasco-Beltrán

state: Bizkaia

address: Bizkaia Science & Technology Park

country: Spain

institute: CIC bioGUNE

summary: We used BD Rhapsody™/scRNA-seq to analyze the heterogeneity of lymphocytes in brain metastasis

overall_design: The immune fraction of brains from intracranially injected mice with B16/f10-BrM cell line (with or without STAT3 inhibition) was sorted to select the target population (DAPI-CD45+CX3CR1-) or separated by magnetic beads using mouse CX3CR1 positive seletion and the negative fraction was analyzed using BD Rhapsody™/scRNA-seq.

first: Borja

last: Jiménez-Lasheras

first: Leire

last: Egia-Mendikute

first: Ana

last: de Pablos-Aragoneses and

first: Neibla

last: Priego


title: Ribo-Seq in Polr3D-/+ mutants

summary: Ribo-Seq, whole female fruit flies, Polr3D heterozygous mutant versus wild-type .

overall_design: Tissues were immediately frozen in liquid nitrogen and ground to power in liquid nitrogen with a mortar and pestle, then dissolved in 400μL of lysis buffer


title: Balancing the CKS1/CKS2 proteostasis axis is critical to maintain hematopoietic stem cell function

first: William

last: Grey

address: 1 Midland Road

institute: Francis Crick Institute

summary: Investigation of the roles of CKS1 and CKS2 in murine haematopoiesis.

overall_design: Bulk RNA sequencing of LSK cells from WT, Cks1b-/- and Cks2-/- 8-week-old mice.

first: Miriam

last: Llorian Sopena


title: Pol III transcribed genes, RNA-Seq, female fruit fly, midgut

summary: RNA-Seq of small (Pol III-transcribed) RNAs in midguts of female fruit flies, Pol III heterozygous mutant versus wild-type


title: Impaired wound healing in Snhg26 knockout mice

city: Nanjing

first: Dongqing

last: Li

address: No.12, Jiangwangmiao Street, Xuanwu District, Nanjing, China

institute: Hospital for Skin Diseases, Institute of  Dermatology, Chinese Academy of Medical Sciences &  Peking Union Medical College

summary: The wounds were made on the back skin of Snhg26 knockout (KO) and wild type (WT) mice. The wound edge and skin tissue were collected and the epidermis were separated by incubate the tissue  with dispaseII. Total RNA was extracted from the epidermis. The global transcriptome analysis of the epidermis were performed by using Affymetrix arrays.

overall_design: Gene expression profiling of the epidermis from the skin and wound edge of Snhg26 KO (n = 3) and WT mice (n = 3) was carried out by using human Clariom™ S assay (ThermoFisher Scientific) at the Bioinformatics and Expression Analysis (BEA) core facility at Karolinska Institutet.

first: Ning

last: Xu Landén


title: Trained immunity is facilitated by T cell-induced CD40-TRAF6 signaling [RNA-seq]

city: Nijmegen

first: Joost

last: Martens

state: Nederland

address: Geert Grooteplein 28

department: RIMLS

country: Netherlands

institute: Radboud University

summary: Trained immunity refers to epigenetic and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to a heightened response to secondary stimuli. Although this innate immune memory contributes to host defense against infections, it can also have maladaptive consequences in a broad range of immune-driven conditions. During the past decade, we have gained an increasingly better understanding of the molecular regulation of trained immunity, but the role of adaptive immune cells in modulation of trained immunity remains largely unknown. Here, we show that T cell interactions contribute to the induction of trained immunity in monocytes and that this mediated by CD40-TRAF6 signaling. Specifically, we found that inhibiting CD40-TRAF6 signaling prevents the induction of functional, transcriptomic, metabolic and epigenetic changes associated with trained immunity. Apart from detailed in vitro studies, we inhibited CD40-TRAF6 signaling in vivo in myeloid cells in a murine heart transplantation model which resulted in prolonged allograft survival. When the animals were additionally treated with costimulatory blockade by CTLA4-Ig, we observed immunological tolerance to the graft. Combined, our study reveals that trained immunity induction is regulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells, and that this signaling can be efficiently leveraged for therapeutic purposes.

overall_design: RNA sequencing on monocytes that are treated with RPMI (ctrl), HKCA or HKCA together with CD40-TRAF6i

first: Maaike

last: Jacobs

middle: H

first: Raphaël

last: Duivenvoorden


title: Transgenic overexpression of the miR-200b/200a/429 cluster prevents mammary tumor development in MTB-TAN mice

city: Guelph

first: Roger

last: Moorehead

address: 50 Stone Rd E

department: Biomedical Sciences

country: Canada

institute: University of Guelph

summary: We have previously shown that transgenic overexpression of the miR-200b/200a/429 cluster prevents mammary tumor development in MTB-IGFIR mice. In this study we evaluated whether the miR-200b/200a/429 cluster could also prevent mammary tumor development from a different oncogene, namely Neu/Erbb2. We found that transgenic overexpression of Neu/Erbb2 in MTB-TAN mice induce rapid mammary tumor development and co-overexpression of the miR-200b/200a/429 cluster with Neu/Erbb2 completely prevent mammary epithelial transformation and tumor development

overall_design: RNA was extracted from mammary gland from 55 day old control, MTB-TAN (overexpress Neu), and MTB-TANba429 (overexpress both Neu and miR-200b/200a/429 cluster) mice as well as mammary tumors from MTB-TAN mice


title: Keratinocyte response to SNHG26 silencing

summary: Analysis the transcriptome of keratinocyte after SNHG26 silencing with or without TNFa treatment for 3 hours. SNHG26 is a conseved lncRNA which is upregulated in the epidermis of human and mouse acute wound. Results of this profiling provide insight into the biological role of SNHG26 in keratinocytes.

overall_design: Human adult primary keratinocytes  were cultured in Epilife medium supplement with Human Keratinocyte Growth Supplement and 1X Penicillin-Streptomycin. 10nM SNHG26-ASO or Ctrl_ASO was transfected for 24 hours followed by TNFa stimulation for another 3 hours. The total RNA was extracted by miRNeasy mini Kits (Qiagen) following the manufacture instruction. The transcriptome profiling of keratinocytes was performed using Affymetrix Clariom S microarray.


title: Enhanced detection of RNA modifications with high-accuracy nanopore RNA basecalling models

city: Barcelona

first: Gregor

last: Diensthuber

state: Barcelona

address: C/ Dr. Aiguader, 88

department: Genome Biology

institute: Center for Genomic Regulation (CRG)

summary: Chemical RNA modifications, collectively referred to as the ‘epitranscriptome’, have been intensively studied during the last years, largely facilitated by the use of next-generation sequencing technologies. Recent efforts have turned towards the nanopore direct RNA sequencing (DRS) platform, as it allows simultaneous detection of diverse RNA modification types in full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling ‘errors’ in DRS datasets, m6A modifications produce very modest ‘errors’, limiting the applicability of this approach to sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully-unmodified in vitro synthetic sequences, increase the ‘error’ signal of m6A modifications, leading to enhanced detection of RNA modifications even at lower stoichiometries. We then show that the use of these models enhances the detection of RNA modifications on previously published in vivo human samples, using third-party softwares for the detection of RNA modifications. Moreover, our work provides a novel RNA basecalling model that shows a median accuracy of 97%, compared to previously available RNA basecalling models that show 91% accuracy. Notably, this increase in accuracy does not only lead to improved detection of RNA modifications, but also enhanced mappability of RNA reads, which becomes more evident in the case of short RNA reads (50% increase). Altogether, our work stresses the importance of using fully unmodified RNA sequences for training RNA basecalling models, and how the use of different basecalling models can significantly affect the detection of RNA modifications and read mappability.

sample_organism:
Arabidopsis thaliana
Saccharomyces cerevisiae
Xenopus laevis
Homo sapiens
Mus musculus
synthetic construct

overall_design:
To compare error-signatures produced by dRNA-seq  across three basecalling models (default, IVT and SUP) and seven RNA modifications types (m6A, m5C, hm5C, ac4C, Ψ, m1Ψ, and m5U) we sequenced fully modified and unmodified in vitro construct. These 'curlcake' sequences contain ever possible 5-mer (n = 1024) in multiple broader sequence contexts (median occurence per 5-mer, n = 10). To provide information on the effect of varying stoichiometries on the modificatoin signature of m6A we additionally sequenced curlcakes generated with the following amount of incorporated m6A (12.5%, 25%, 50% and 75% modified).
Subsequently we tested whether our novel basecalling models can lead to an imporved detection of m6A sites on in vivo data. We used publicly availabe dRNA-seq data of wildtype and Mettl3-/- HEK293T cells present in biological triplicates which we basecalled using three different models (default, IVT and SUP). To identify m6A modified sites we performed pairwise-comparisons using eligos2 between widltype and knockout samples
To compare read accuracy and mapping ability of the three tested basecalling models, we used publicly available dRNA-seq data for several model species (H. sapiens, M. musculus, X. laevis, S. cerevisiae and A. thaliana)
We tested the ability of two basecalling models (default and SUP) to detect m1Ψ in a synthetic mRNA vaccine

last: Dienstuber

first: Leszek

last: Pryszcz

first: Laia

last: Llovera

first: Morghan

last: Lucas

first: Anna

last: Delgado-Tejedor

first: Sonia

last: Cruciani

first: Oguzhan

last: Begik

first: Eva

last: Novoa


title: In vivo contribution of Cyp24a1 promoter vitamin D response elements

status: Public on Aug 25 2024

first: Mark

middle: B

last: Meyer

state: WI

address: 1415 Linden Dr.

department: Nutritional Sciences

summary: CYP24A1 is a multifunctional, P450 mitochondrial enzyme that catabolizes the vitamin D hormone (calcitriol, 1,25(OH)2D3), its precursor (calcifediol, 25(OH)D3), and numerous vitamin D metabolites. In the kidney, Cyp24a1 is induced by 1,25(OH)2D3 and fibroblast growth factor 23 (FGF23) and potently suppressed by PTH to control the circulating levels of 1,25(OH)2D3. Cyp24a1 is controlled by a pair of promoter proximal (PRO) vitamin D response elements (VDREs) that are aided by distal, downstream (DS) enhancers. The downstream 1 region of Cyp24a1 (DS1) enhancer is kidney-specific and responsible for PTH and FGF23 actions, and the downstream 2 region of Cyp24a1 enhancer responds to 1,25(OH)2D3 in all tissues. Despite this knowledge, in vivo contributions of the PRO VDREs to basal expression, FGF23 activation, and PTH suppression of Cyp24a1 remain unknown. In this study, we selectively mutated the PRO VDREs in the mouse to address these questions. We found mutation of the VDREs leads to a dramatic loss of VDR occupancy, a reduction of 1,25(OH)D3-induced kidney Cyp24a1 expression, and near elimination of intestinal Cyp24a1 induction. FGF23 induction of Cyp24a1 was reduced but not eliminated and still showed a synergistic increase with 1,25(OH)2D3. PTH suppression of Cyp24a1 was unchanged, despite minor reductions in total for phosphorylated cAMP-response element binding protein occupancy. Finally, VDR recruitment was dramatically reduced across the DS enhancers in the Cyp24a1 locus. Taken together, our data suggest a cooperative relationship between the DS and PRO enhancers in the regulation of Cyp24a1 by 1,25(OH)2D3 and FGF23 and points to the DS1 region as a crucial basal switch for Cyp24a1 activity that further defines the interconnected genomic control in vitamin D catabolism.

overall_design: Chromatin Immunoprecipitation sequencing (ChIP-seq) of VDR and phosphorylated CREB from both mouse kidney and intestine tissues from both wildtype mice and C24-V1V2 mutation mice. Input sample from WT kidney is accession: GSM3898546.


title: Effect of U2AF1-S34F mutation in mouse neutrophils

city: Denver

first: Scott

last: Alper

state: CO

address: 1400 Jackson St.

institute: National Jewish Health

summary: To investigate the effect of the MDS-associated U2AF1-S34F mutation on gene expression and pre-mRNA splicing in mouse neutrophils, we performed RNA-seq on RNA isolated from bone marrow neutrophils expressing either U2AF1-S34F or U2AF1-WT transgenes

overall_design: Comparative gene expression profiling and alternative splicing analysis


title: An aberrant immune-epithelial progenitor niche drives post-viral lung sequelae [mouse]

first: Harish

last: Narasimhan

state: Virginia

address: 345 Crispell Drive

summary: Respiratory viral infections are being increasingly recognized not just for their acute impact but also as potential triggers of long-term health conditions. The recent COVID-19 pandemic in particular has highlighted the prevalence of this phenomenon, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), which has rapidly evolved into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or impaired organ recovery post infection. Yet, the precise mechanisms linking non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC. Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. CD8+ T cell derived IFN-γ and TNF stimulated local macrophages to chronically release IL-1β, resulting in the abnormal accumulation of dysplastic epithelial progenitors and lung fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1β after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate an aberrant immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

overall_design: Lungs from 4 aged C57BL/6 mice infected with influenza were treated with control (S1A, S2A) or αCD8 (S1D, S2D) antibody prior to collection and Visium analysis at 60 days post infection

first: Sun

last: Jie

first: Narasimhan

last: Harish


title: A mouse model of human coronavirus HCoV-NL63 infection: comparison with rhinovirus-A1B

city: Ann Arbor

first: Marc

last: Hershenson

state: MI

address: 1150 W. Medical Center Drive

department: Department of Pediatric Pulmonology

institute: Michigan Medicine

summary: We establish a mouse model of HCoV-NL63 and compare outcomes with a model of rhinovirus (RV)-A1B.  Transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell lysate,  100,000 TCID50 units HCoV-NL63, or 1,000,000 PFU RV-A1B.  Lungs were assessed for host gene expression by next generation sequencing.  RNAseq showed increases in IFN-α, IFN-β, IFN-λ and many IFN-stimulated genes (ISGs) in NL63-infected mice.  In addition to ISGs, RV-A1B induced expression of IFN-γ, IL-1β, NLRP3, TLR2 and C-X-C chemokines. We established a mouse model of HCoV-NL63 replicative infection characterized by expression of ISGs but fewer pro-inflammatory genes compared to RV-A1B.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for B6.Cg-Tg(K18-ACE2)2Prlmn/J mice treated with RVA1B or HCoV-NL63.

first: J

middle: K

last: Bentley

first: Jordan

last: Kreger

first: Haley

last: Breckenridge

first: Shilpi

last: Singh

first: Yiran

last: Li

first: Susan

last: Baker

first: Carey

middle: N

last: Lumeng


title: An aberrant immune-epithelial progenitor niche drives post-viral lung sequelae [human]

overall_design: Lungs from 3 human PASC-PF patients and 2 control patients were collected for Visium analysis


title: Transcriptome-wide mapping of RNA:protein interactions of GFP-RS2Z35 and GFP-RS2Z36 in tomato (Solanum lycopersicum) leaves.

city: Frankfurt am Main

first: Michaela

last: Müller-McNicoll

address: Max-von-Laue-Str. 13

institute: Goethe University Frankfurt

summary: We performed iCLIP2 in tomato leaves stably expressing GFP-tagged RS2Z35 or RS2Z36.

overall_design: 3 biological replicates were subjected to the iCLIP2 procedure with UV-crosslinking at 254 nm at 750 mJ/cm2. For IPs GFPTrap magnetic agarose used. No UV treatment served as negative control.

first: Sotirios

last: Fragkostefanakis

first: Remus

middle: R

last: Rosenkranz

first: Anke

last: Busch


title: Trained immunity is facilitated by T cell-induced CD40-TRAF6 signaling [ChIP-seq]

first: Joost

last: Martens

overall_design: ChIP sequencing on monocytes that are treated with RPMI (ctrl), HKCA or HKCA together with CD40-TRAF6i

first: Maaike

last: Jacobs

first: Raphaël

last: Duivenvoorden


title: Lysosomal catabolic activity governs the exit of the murine totipotent 2-cell state by silencing early-embryonic retrotransposons [CUT&Tag]

city: HangZhou

first: hao

last: wu

address: YuHang street

institute: Zhejiang University

summary: During the development in mouse early embryos, a subset of retrotransposons and genes are transiently expressed in the totipotent 2-cell embryos. These 2-cell-embryo-asscoiated (referred to as 2C) transcripts rapidly shut down their expression beyond the late 2-cell stage of embryos, and this timely shutdown governs the embryo to exit from the 2-cell stage. However, mechanisms regulating this wave of shutdown are not fully understood. In this study, we established a novel connection between lysosomal catabolic activity and 2C transcripts in regulating the exit of totipotent 2-cell state by using both in vitro 2-cell-like cells model and mouse embryos. Our results showed that the activation of lysosomal catabolism in mouse embryos governed the embryo to exit from the 2-cell state by suppressing the expression of 2C transcripts. Mechanistically, we found that lysosomal catabolic activity modulated 2C transcripts by inactivating transcriptional factors Tfe3/Tfeb and abolishing their transcriptional activation on 2C retrotransposons, MERVL/MT2-Mm. Collectively, our study elucidates the function of lysosomal activity in orchestrating transcriptomic landscape and early development in mouse embryos and reveals an unanticipated layer of transcriptional control on early-embryonic retrotransposons from lysosomal signaling.

overall_design: CUT&Tag assay for Flcn knockout mESCs.

first: Hao

last: Wu

first: Lanrui

last: Cao

first: Xinpeng

last: Wen

first: Xudong

last: Fu


title: Lysosomal catabolic activity governs the exit of the murine totipotent 2-cell state by silencing early-embryonic retrotransposons

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

overall_design: Refer to individual Series


title: Lysosomal catabolic activity governs the exit of the murine totipotent 2-cell state by silencing early-embryonic retrotransposons [RNA-seq]

overall_design: RNA-seq for Vacuolin-1 treated and Flcn Knock down mESCs. RNA-seq for Vacuolin-1 treated and Flcn Knock down embryos.


title: Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [sci-RNA-seq]

status: Public on Aug 23 2024

city: Seattle

first: Sergei

last: Doulatov

state: Washington

address: 1959 NE Pacific St K259

institute: University of Washington

summary: Hematopoietic stem cells (HSCs) arise in mid-gestation from a specialized hemogenic endothelium (HE). In this process, HE cells undergo a unique fate change termed endothelial-to-hematopoietic transition, or EHT. While induced pluripotent stem cells (iPSCs) give rise to HE with robust hemogenic potential, the generation of bona fide HSCs from iPSCs remains a challenge. Here, we map single cell dynamics of EHT during embryoid body differentiation from iPSCs and integrate it with human embryo datasets to identify key transcriptional differences between in vitro and in vivo cell states. We further map ligand-receptor interactions associated with differential expression of developmental programs in the iPSC system. We found that the expression of endothelial genes was incompletely repressed during iPSC EHT. Elevated FGF signaling by FGF23, an endothelial pathway ligand, was associated with differential gene expression between in vitro and in vivo EHT. Chemical inhibition of FGF signaling during EHT increased HSPC generation in the zebrafish system, while an FGF agonist had the opposite effect. Consistently, chemical inhibition of FGF signaling increased hemogenic output from iPSCs. In summary, we map the dynamics of EHT from iPSCs at single cell resolution and identify ligand-receptor interactions that can be modulated to improve iPSC differentiation protocols. We show, as proof of principle, that chemical inhibition of FGF signaling during EHT improves hematopoietic output in zebrafish and the iPSC system.

overall_design: iPSCs were formed into embryoid bodies (day 0) and treated with 100ng/ml hSCF, 100ng/ml hFLT3, 50ng/ml hG-CSF, 25ng/ml IL-6 and 10ng/ml IL-3 starting on day 1 and throughout the rest of any given experiment. Unsorted cells from each time point (days 7, 8, 11, 18 and 21 of differentiation) were used for single-cell combinatorial indexing RNA-seq (sci-RNA-seq) samples.  CD34+, CD43+ or CD45+ were sorted using FACS for the 10X 3' sc-RNA-seq day 8 sample.

first: Rachel

last: Wellington

first: Xiaoyi

last: Cheng

first: Clyde

last: Campbell

first: Cole

last: Trapnell

first: Raquel

last: Espin-Palazon

first: Brandon

last: Hadland


title: Towards a CRISPR-Based Mouse Model of Vhl-deficient Clear Cell Kidney Cancer: Initial Experience and Lessons Learned

status: Public on Aug 24 2024

city: Bethesda

first: Christopher

middle: James

last: Ricketts

state: Maryland

address: Building 10

department: Urologic Oncology Branch

institute: National Cancer Institute

summary: CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models.  Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC).  Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective.  Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors.  An AAV targeting  Vhl, Pbrm1, Keap1 and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard of care agent, axitinib.  RNAseq analysis was utilized to evaluate the expression profile produced by the targeted loss of  Vhl, Pbrm1, Keap1 and Tsc1 in these mouse tumors. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs,  are HIF2-independent.

overall_design: RNA was extracted from 10 seperate tumor that were derived from 10 sperate C57BL/6 mice that were directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL, Pbrm1, Keap1 and Tsc1. RNA was also extracted from normal kidney regions from the smae mice.  RNAseq was performed on all 20 RNAs and the differential expression profile between the tumors and normal samples are evaluated.

first: Laura

last: Stransky

last: Schmidt

first: William

middle: G

last: Kaelin Jr


title: Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [10X 3' sc-RNA-seq]


title: Pericyte Phenotype Switching Alleviates Immunosuppression and Sensitizes Vascularized Tumors to Immunotherapy in Preclinical Models

city: Perth

first: Alistair

middle: R. R.

last: Forrest

state: WA

address: 6 Verdun Street, Nedlands

country: Australia

institute: Harry Perkins Institute of Medical Research, The University of Western Australia

summary: The efficacy of T cell-based immunotherapies is limited by the immune-suppressive tumor environment mediated by hypoxia, arising from dysfunctional blood vessels. Normalizing the tumor vascular bed can translate into an immune-supportive microenvironment. Here, we show that targeting pericytes to achieve durable vessel normalization can improve immunotherapy outcomes. Specifically, we identified regulator of G protein signaling 5 (RGS5) as a master regulator of tumor pericyte differentiation. Loss of RGS5 function changes the tumor microenvironment by inducing pericyte quiescence and maturity via Rho kinase activation and expression of contractile proteins. The mature pericyte state affords highly sustained vascular changes, which profoundly facilitates anti-tumor immune activity. Importantly, we show that selective low dose therapeutics can induce pericyte maturity and contractility, which mimics genetic RGS5 loss in both mouse and human tumors. Low dose drug therapy durably changes the tumor microenvironment without inducing adaptive resistance, rendering malignant cells more sensitive to immunotherapies. Our findings create a new approach with highly translatable opportunities to improve the outcomes of immune combination therapies.

overall_design: Tumors from 27 week-old RIP1-Tag5 mice were harvested and analysed using scRNA-seq.

first: Zhi-Jie

last: Li

first: Bo

last: He

first: Alice

last: Domenichini

first: Jiulia

last: Satiaputra

first: Kira

last: Wood

first: Abate

last: Bashaw

first: Lisa

last: Nilsson

first: Devina

last: Lakhiani

first: Ji

first: Bastow

last: R Edward

first: Anna

last: Johansson-Percival

first: Elena

last: Denisenko

first: Rafael

last: Carretero

first: Günter

middle: J

last: Hämmerling

first: Jonas

first: Gabriel

middle: Y

last: Lee

first: Ruth

last: Ganss


title: Super-enhancer H3K27ac CUT& Tag sequencing of six human gastric cancer tissue samples

city: Fuzhou, Fujian, China

first: Jie

last: Liu

address: Shengli Clinical Medical College of Fujian Medical University, Department of Endoscopy, Fujian Provincial Hospital, Fuzhou, China

institute: Fujian Medical University

summary: CUT& Tag was performed using H3K27ac antibody to identify the super-enhancers of GC with different growth patterns through ROSE algorithm. Comparison of SE-driven oncogenes in infiltrative and expanding GC was carried out to screen key signatures and candidate genes.

overall_design: Six GC samples were collected, and H3K27ac CUT&Tag sequencing was performed for the first time to identify of GC SEs


title: Comparative gene expression analysis in OCI-LY8 isogenic cell lines with SETD1B mutation or WT control.

city: New York

first: Bic

last: MSKCC

state: NY

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary: The translocation t(14;18) activates BCL2 and is considered the initiating genetic lesion in most follicular lymphomas (FL). Surprisingly, FL patients fail to respond to the BCL2 inhibitor, Venetoclax. We show that mutations and deletions affecting the histone lysine methyltransferase SETD1B (KMT2G) occur in 7% of FLs and in 16% of diffuse large B cell lymphomas (DLBCL). Deficiency in SETD1B confers striking resistance to Venetoclaxand to an experimental MCL-1 inhibitor. SETD1B also acts as a tumor suppressor andcooperates with loss ofKMT2Din lymphoma development in vivo. Consistently, loss of SETD1B in human lymphomas typically coincides with loss of KMT2D. Mechanistically, SETD1B is required for the expression of several pro-apoptotic BCL2 family proteins. Conversely, inhibitors of the KDM5 histoneH3K4demethylases restore BIM and BIK expression and synergize with Venetoclax inSETD1B-deficient lymphomas.

overall_design: To investigate the specific role of SETD1B mutations and the cooperative function of SETD1B and KMT2d duble mutant, we stablish OCI-LY19 (KMT2D mutant) isogenic cell lines with using sgRNAs tageting SETD1B or an sgNTC(control).

first: Shenqiu

last: Wang

first: Jiayu

last: Wen

first: Alexander

last: Gorelick

first: Ana

last: Portelinha


title: H3K5me3 modifications in OCI-LY19 cells with and without SETD1B mutations.

overall_design: Chromatin Immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K5me3 in OCI-LY19 cells with and without mutations in SETD1B.


title: Pol III transcribed genes, RNA-Seq, C.elegans

first: Nazif

last: Alic

address: Gower St

summary: RNA-Seq of small (Pol III-transcribed) RNAs in worms, Pol III RNAi versus control

overall_design: RNAs isolated, size selected and demethylated before samples sent to Glasgow Polyomics. RNAseq

first: Yasir

last: Malik

first: Yavuz

last: Kulaberoglu

first: Gillian

last: Borland

first: Colin

last: Selman

first: Jennifer

last: Tullet


title: Enhancer-driven cell type comparison reveals similarities between the mammalian and avian telencephalon

city: Leuven

first: Stein

last: Aerts

address: Herestraat 49 PO Box 602

department: Center for Brain and Disease Research

country: Belgium

institute: VIB-KU Leuven

summary: Combinations of transcription factors govern the identity of cell types, which is reflected by genomic enhancer codes. We utilized deep learning to characterize these enhancer codes and devised three novel metrics to compare cell types in the telencephalon between mammals and birds. To this end, we generated single-cell multiome and spatially-resolved transcriptomics data of the chicken telencephalon. Enhancer codes of orthologous non-neuronal and GABAergic cell types show a high degree of similarity across vertebrates, while excitatory neurons of the mammalian neocortex and avian pallium exhibit varying degrees of similarity. Enhancer codes of avian mesopallial neurons are most similar to those of mammalian deep layer neurons. With this study, we present generally applicable deep learning approaches to characterize and compare cell types solely based on genomic sequences.

overall_design: 15 days domestic chicken (broiler) were used for the experiments. Coronal sections of the telencephalon were dissected and analyzed with Nova-ST spatial transcriptomics

first: Nikolai

last: Hecker

first: Niklas

last: Kempynck

first: David

last: Mauduit

first: Darina

last: Abaffyova

first: Roel

last: Vandepoel

first: Sam

last: Dieltens

first: Lars

last: Borm

first: Ioannis

last: Sarropoulus

first: Carmen

last: Bravo Gonzalez-Blas

first: Julie

last: De Man

first: Kristofer

last: Davie

first: Elke

last: Leysen

first: Gert

last: Hulselmans

first: Lynette

last: Lim

first: Joris

last: De Wit

first: Valerie

last: Christiaens

first: Suresh

last: Poovathingal


title: Comparative gene expression analysis in OCI-LY19 isogenic cell lines with SETD1B mutation or KMT2D+SETD1B double mutants.

first: Bic

last: MSKCC

first: Shenqiu

last: Wang

first: Jiayu

last: Wen

first: Alexander

last: Gorelick

first: Ana

last: Portelinha


title: Inter-organ transmission of hepatocellular senescence induces multi-organ dysfunction through the TGFbeta signalling pathway

city: Glasgow

first: Thomas

last: Bird

address: Switchback Road, Bearsden

department: CRUK Scotland Institute

institute: Beatson Institute

summary: Cellular senescence is associated with aging but also impacts various physiological and pathological processes such as embryonic development and wound healing. Factors secreted by senescent cells can affect their microenvironment, including local spreading of senescence. Acute severe liver disease is associated with hepatocyte senescence and frequently progresses to multi-organ failure. Why the latter occurs is poorly understood however, the presence of hepatic senescence is associated with poor prognosis and extrahepatic organ failure in acute liver disease. Here, using genetic mouse models of hepatocyte-specific senescence, we demonstrate senescence development in extrahepatic organs and associated organ dysfunction in response to liver senescence. In patients with acute indeterminate hepatitis, the extent of hepatocellular senescence predicts the occurrence of extrahepatic dysfunction, need for liver transplantation and mortality. We identify the Transforming Growth Factor Beta (TGFbeta) pathway as a critical mediator of systemic spread of senescence and TGFbeta inhibition blocks senescence transmission to other organs preventing renal dysfunction. Our results highlight the systemic consequences of organ-specific senescence which, independent of aging, contributes to multi-organ dysfunction.

overall_design:
An adeno-associated viral (AAV) vector system is used to express the mutant KRasG12D in the hepatocytes of mice.
Bulk RNA-Seq was used to determine the transcriptional differences between control and mutant KRasG12D. 4 AAV8-Cre2 controls, 4 AAV8-Cre2 treatments , 4 AAV8-Cre4 controls, 4 AAV8-Cre4 treatments, and 4 untreated controls from kidney tissue were used. The samples were split over two sequence runs 180804 and 180805.
The TruSeq Stranded mRNA kit was used and the samples sequenced on a NextSeq 500. Raw fastq.gz where quality trimmed with FastP and aligned to the GRCm38.92 genome with HiSat2. The reads where summarized using Feature Counts.

first: Christos

last: Kiourtis

first: Maria

last: Terradas-Terradas

first: Lucy

last: Gee

first: Stephanie

last: May

first: Anastasia

last: Georgakapoulou

first: Amy

last: Collins

first: Miryam

last: Müller

first: Eoin

middle: D

last: O'Sullivan

first: David

middle: P

last: Baird

first: Mohsin

last: Hassan

first: Ee

last: Hong Ta

first: Cornelius

last: Engelmann

first: Fausto

last: Andreola

first: Ya-Ching

last: Hsieh

first: Lee

last: Reed

last: Borthwick

first: Colin

last: Nixon

first: William

last: Clark

first: Robin

last: Shaw

first: Peter

last: Hanson

last: Sumpton

first: Gillian

last: Mackay

first: Toshi

last: Susuki

first: Arafath

last: Najumudeen

first: Andrew

last: Campbell

first: Simon

middle: T

last: Barry

first: Alberto

last: Quaglia

first: Christopher

last: Morris

first: Fiona

middle: E

last: LeBeau

first: Owen

last: Sansom

last: Ferenbach

first: Kristina

last: Kirschner

first: Rajiv

last: Jalan

last: Oakley


title: PPARβ/δ-orchestrated metabolic reprogramming supports the formation and maintenance of memory CD8+ T cells [scRNA-seq]

city: Epalinges

first: Ping-Chih

last: Ho

address: Ch. des Boveresses 155

country: Switzerland

institute: University of Lausanne

summary: The formation of antigen-specific memory CD8+ T cells is one of the most important features of the adaptative immune system, allowing the establishment of long-term protection against secondary infections. Although emerging evidence suggests that metabolic reprogramming is crucial for memory T cell differentiation and survival, the underlying mechanisms that drive metabolic rewiring needed for memory T cells remain unclear. Here, we found that the nuclear receptor peroxisome proliferator-activated receptor-beta/delta (PPARβ/δ) was upregulated to instruct the metabolic reprogramming, including downregulation of aerobic glycolysis and the promotion of oxidative metabolism and fatty acid oxidation, that occurs during the transition toward the establishment of central memory CD8+ T cells. Mechanistically, the exposure to interleukin-15 (IL-15) and expression of T cell factor 1 (TCF1) could coordinately activated the PPARβ/δ pathway during acute viral infection and chronic antigen exposure contexts, counteracting apoptosis induced by antigen clearance and metabolic stress. Together, our work indicates that PPARβ/δ is a master metabolic regulator orchestrating the metabolic reprogramming required for the establishment of a metabolic profile favorable for T cells longevity.

overall_design: 10K P14 WT and PPARb deficient CD8+ T cells were transferred in B6 host which were infected the next next with LCMV Armstrong. Transferred P14 CD8+ T cells were sorted from spleens at d15 after Arm infection and prepared and then sequenced using the Chromium Next GEM Single Cell 3ʹ kit from 10x Genomics

first: Alessio

last: Bevilacqua

first: Yu-Ming

last: Chuang

first: Nabil

last: Rahiman

first: Xin

last: Xie


title: Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos

city: Columbia

first: Wipawee

last: Winuthayanon

state: Missouri

address: 1030 Hitt Street

department: OB/GYN & Women's Health

institute: University of Missouri

summary: The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner.

overall_design: Adult C57B6/J wild-type female mice were mated with either vasectomized or proven breeder males. In some cases, females were superovualted with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrpin (hCG) before mating. The oviduct tissues were collected at 0.5, 1.5, 2.5, and 3.5 days post coitus (dpc) or in some case days of pseudopregnancy (dpp). The oviducts were dissected into infundibulum+ampulla (IA) or isthmus+uterotubal junction (IU) regions. Samples were then processed for bulk RNA-sequencing or single cell RNA-sequencing.

first: Ryan

middle: M

last: Finnerty

first: Daniel

last: Carulli

first: Akshata

last: Hedge

first: Yanli

last: Wang

first: Frimpong

last: Baodu

first: Sarayut

first: Jianlin

last: Cheng


title: Single-cell transcriptional profiles of the atrioventricular canal of 1-month-old zebrafish treated with DMSO or IWR-1-endo

city: Yamagata

first: Ayano

last: Chiba

state: Yamagata

address: 2-2-2 Iida-Nishi

department: Department of Pharmacology

country: Japan

institute: Yamagata University

summary: To identify the crosstalk between Wnt/β-catenin signaling activated cardiomyocytes (β-cat ON CMs) and coronary endothelial cells (coECs), we performed single-cell RNA sequencing on all the cells of the atrioventricular canal (AVC) at 1 month, which contains newly formed coronary vessels. To identify the extracellular ligands regulated by Wnt/β-catenin signaling, we also collected AVC from fish treated with DMSO or IWR1-endo, an inhibitor of Wnt/β-catenin signaling. Our analyses indicated that Fgf and/or Sema signaling are potential candidates for mediating the crosstalk between β-cat ON CMs and coECs.

overall_design: We treated 1-month-old Tg(cryaa:EGFP, myl7:GAL4db-TCFΔC);(5xUAS:NTR-mCherry) fish with DMSO or 10 μM IWR1-endo for 48 hours. Hearts were resected from the fish treated with MTZ or DMSO, respectively. We isolated AVCs from hearts and dissociated them into single cells. Barcoded single-cell cDNA libraries were prepared using the Chromium Next GEM single cell 3’reagent kits v3.1 and sequenced using Illumina NextSeq 500.

first: Manabu

last: Shirai

first: Takuya

last: Yamamoto

first: Hiroyuki

last: Nakajima

first: Naoki

last: Mochizuki


title: HIV-1 Vpr-induced DNA damage activates NF-κB through ATM-NEMO independent of cell cycle arrest

city: Los Angeles

first: Oliver

middle: I.

last: Fregoso

state: CA

address: 615 Charles E Young Dr S, Los Angeles CA 90095

department: Microbiology, Immunology and Molecular Genetics

institute: University of California Los Angeles

summary: Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through DNA damage, cell cycle arrest, the degradation of host proteins, and both the activation and repression of DDR signaling. Vpr also alters host and viral transcription; however, the connection between Vpr-mediated DDR modulation and transcriptional activation remains unclear. Here, we determined the cellular consequences of Vpr-induced DNA damage using Vpr mutants that allow us to separate the ability of Vpr to induce DNA damage from cell cycle arrest and other DDR phenotypes including host protein degradation and repression of DDR. RNA-sequencing of cells expressing Vpr or Vpr mutants identified that Vpr alters cellular transcription through mechanisms both dependent and independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), Vpr-induced DNA damage activates the ATM-NEMO pathway and alters cellular transcription via NF-κB/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-κB transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional reprogramming can occur during early and late stages of viral replication. Together, our data identifies a mechanism by which Vpr activates NF-κB through DNA damage and the ATM-NEMO pathway, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral transcription and replication.

overall_design: To determine if Vpr WT and mutants alter the cellular transcriptome, we performed RNA-seq on U2OS cells infected with recombinant adeno-associated virus (rAAV) expressing either Vpr WT or Vpr mutants S79A, H71R and Q65R. U2OS cells treated with empty vector rAAV were used as negative vehicle control, and untreated U2OS cells served as negative control. Total RNA was collected at 36 hours post infection. Experiment was done with technical triplicates. Using the statistical significance of Log2 (1.25), p < 0.01, and FDR < 4.50E-05, We found that Vpr WT, H71R and S79A alters cellular transcription compared to untreated cells. Furthermore, Vpr Q65R and empty vector rAAV treated cells did not alter transcription compared to untreated cells.

first: Carina

last: Sandoval

first: Karly

last: Nisson

middle: I


title: PPARβ/δ-orchestrated metabolic reprogramming supports the formation and maintenance of memory CD8+ T cells

first: Ping-Chih

last: Ho

overall_design: 10K P14 WT and PPARb deficient CD8+ T cells were transferred in B6 host which were infected the next next with LCMV Armstrong. After 28 days, the spleens were collected and the central memory CD8+ transferred cells WT and KO were sorted (CD62l+ CD127+). RNA was extracted using the RNeasy® Mini Kit (Qiagen) following the instructions of the manufacturer. Bulk RNA-seq was then performed.

first: Alessio

last: Bevilacqua

first: Yu-Ming

last: Chuang


title: Poly-GR impairs translation elongation and increases ribotoxic stress response in C9ORF72-ALS/FTD

city: Baltimore

first: Shuying

last: Sun

state: MD

address: Hunterian 205

department: Physiology

institute: jhmi

summary: Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Poly-GR is one of the most toxic dipeptide repeat (DPR) proteins translated from the RNA repeats.  It has been shown to affect protein synthesis, but how this contributes to neurodegeneration is not clear. Here, we found that poly-GR inhibits global translation by perturbing translation elongation. We identified that the transcripts with relatively slow elongation rate tend to be further stalled by poly-GR in iPSC-differentiated neurons. This increases ribosome collision and ZAKα-mediated ribotoxic stress response (RSR), which elevates the phosphorylation of p38  and promotes cell death. Knockdown of ZAKα or pharmacological inhibition of p38 can ameliorate the GR toxicity, and improve the survival of C9ORF72-ALS/FTD patient-derived iPSC-neurons. Our study reveals molecular mechanism of poly-GR mediated toxicity on global translatome, and identifies RSR as a potential therapeutic target for C9ORF72-ALS/FTD.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for i3Neuron cells expressing GFP or GR50 from total input lysates or polysome associated mRNA before and after adding Harringtonine

first: Daoyuan

last: Dong

first: Zhe

last: Zhang

first: Yini

last: Li

first: Shaopeng

first: Malgorzata

last: Latallo

first: Blake

last: Nelson

first: Rong

last: Wu

first: Bin

first: Gopinath

last: Krishnan

first: Fen-Biao

last: Gao


title: Context-Dependent Roles of Mitochondrial LONP1 in Orchestrating the Balance between Airway Progenitor versus Progeny Cells

city: San Diego

first: Le

last: Xu

address: 8018 Regents Road Atp. 301

institute: University of California, San Diego

summary: While all eukaryotic cells are dependent on mitochondria for function, in a complex tissue, which cell type and which cell behavior are more sensitive to mitochondrial deficiency remains unpredictable. Here we show that in the mouse airway, compromising mitochondrial function by inactivating mitochondrial protease gene Lonp1 led to reduced progenitor proliferation and differentiation during development; apoptosis of terminally differentiated ciliated cells and their replacement by basal progenitors and goblet cells during homeostasis; and failed airway progenitor migration into damaged alveoli following influenza infection. ATF4 and the integrated stress response (ISR) pathway are elevated and responsible for airway phenotypes. Such context dependent sensitivities are predicted by the selective expression of Bok, which is required for ISR activation. Reduced LONP1 expression is found in chronic obstructive pulmonary disease (COPD) airways with squamous metaplasia. These findings illustrate a cellular energy landscape whereby compromised mitochondrial function could favor the emergence of pathological cell types.

overall_design: Bulk RNA-seq from embryonic lungs at E13.5, including 3 controls and 4 Shhcre;Lonp1 mutants.  Single cell RNA-seq from purified adult airway epithelial cells of control and Sox2creER;Lonp1;tdTomato. scRNA-seq from control (PBS) and influenza infected lungs at D11 and D20.

first: Chunting

last: Tan

first: Justinn

last: Barr

first: Xin


title: DNA Double-Strand Break Movement in Heterochromatin Depends on the Histone Acetyltransferase dGcn5.

city: Utrecht

first: Aniek

last: Janssen

address: Universiteitsweg 100

institute: University Medical Center Utrecht

summary: Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB repair process. Specifically, DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using locus-specific DSB induction in Drosophila tissues and cultured cells, we identify that histone H3 lysine 9 acetylation (H3K9ac) is enriched at DSBs in heterochromatin but not euchromatin. We find that this enrichment is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatin DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

overall_design: Drosophila Kc 167 cells were transfected with double stranded RNA against target genes to asses the effect of Gcn5 knockdown on DSB repair genes and cell cycle related genes

first: Apfrida

last: Kendek

first: Arianna

last: Sandron

first: Jan-Paul

last: Lambooij

first: Serafin

middle: U

last: Colmenares

first: Severina

last: Pociunaite

first: Iris

last: Gooijers

first: Lars

last: de Groot

first: Gary

middle: H

last: Karpen


title: Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS

first: Shuying

last: Sun

summary: Dysregulation of RNA processing contributes to neurodegenerative diseases, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common cause of both FTD and ALS (C9-FTD/ALS), characterized with aberrant repeat RNA foci in the nucleus and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions in the cytoplasm. Here we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon-skipping and intron retention in human iPSC-derived neurons. Similar alternative splicing changes can be found in patient postmortem tissues. This work identified novel molecular mechanism of global RNA splicing defects by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.

overall_design: To investigate nuclear speckle dysfuntion induced RNA splicing change in human neurons, we knockdown nuclear speckle essential components SON and SRRM2 by transduction with SON ans SRRM2 shRNA in human iPSC-derived i3Neurons.

first: Rong

first: Yingzhi

last: Ye

first: Daoyuan

last: Dong

first: Yini

last: Li


title: Single cell transcriptome level assessment of human platelet-biased hematopoietic stem cells isolated from bone marrow samples obtained from healthy young and old adult donors.

city: Oxford

first: Claus

last: Nerlov

address: John Radcliffe Hospital, University of Oxford

department: MRC Molecular Haematology Unit

institute: Weatheral Institute of Molecular Medicine

summary: Functional heterogeneity of hematopoietic stem cells (HSCs) heterogeneity has been extensively described in mice, but so far not described in human bone marrow (BM). In particular, HSCs biased towards platelet formation have been identified, and shown to be expanded during ageing and implicated as cells-of-origin for essential thrombocythemia (ET). By combining xenografting of molecularly barcoded adult human BM HSCs and high-throughput single cell RNA sequencing we here identify human BM HSCs that are molecularly and functionally platelet-biased. Furthermore, a quantitative comparison of transcriptomes from young and aged BM HSCs showed that both the proportion of platelet-biased HSCs and their level of molecular platelet priming increases with age.

overall_design: Examination of the transcriptome of single cell human bone marrow HSCs (Lin–CD34+CD38–CD90+CD45RA–) from healthy adult young (21-24yo) and old (68-75yo) donors.

first: Bilyana

last: Stoilova

first: Mario

last: Buono

first: Merve

last: Aksöz

first: Niels

middle: A

last: Jacobsen

first: Paresh

last: Vyas


title: Macrophage-expressed coagulation factor 7 promotes adverse cardiac remodeling

city: Mainz

first: Wolfram

last: Ruf

address: Langenbeckstrasse 1

institute: CTH Mainz

summary: Background: Excess fibrotic remodeling leads to cardiac dysfunction in ischemic heart disease and is driven by MAP kinase-dependent transforming growth factor-ß1 (TGF-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is incompletely understood.  Methods: Mice with permanent ligation of the proximal left anterior descending artery (LAD) were used to model ischemic heart disease and analyzed by single cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-factor 7 (F7)-integrin ß1-protease activated receptor (PAR) 2 signaling complex by utilizing genetic mouse models and pharmacological intervention. Results: Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared to controls. Proximity ligation assays of monocytic cells in the infarcted myocardium demonstrated that colocalization of F7 with integrin ß1 was diminished in monocyte/macrophage F7-deficient mice. F7fl/fl CX3CR1Cre relative to littermate control mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells into the infarct zone. Single cell mRNA sequencing of CD45+ cells 3 and 7 days after MI uncovered a trajectory from ischemic myocardium-recruited monocytes to inflammatory TF+/F7+/TREM1+ macrophages. As early as 7 days after MI, macrophage F7-deletion led to an expansion of Olfml3+ macrophages with a reparative phenotype and, conversely, to a reduction of TF+/F7+/TREM1+ macrophages, which were also attenuated in activation-resistant PAR2R38E mice.  To demonstrate the therapeutic potential of inhibiting the TF-F7-PAR2 signaling complex,, we injected a specific monoclonal anti-TF antibody that lacks anticoagulant activity, but interferes with macrophage migration in vitro. Short-term treatment from day 1-5 after non-reperfused MI improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI. Conclusions: Extravascular TF-F7-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

overall_design: Cd45+ selected cells from F7fl/fl CX3CR1Cre and F7fl/fl WT, isolated from the infarcted myocardium at different timepoints post MI (permanent ligation) were single cell sequenced on the Rhapsody platform.

first: Venkata

last: Garlapati

first: Jens

last: Posma

first: Tobias

last: Bopp

first: Philip

last: Wenzel


title: Antibody Sequence Determinants of Viral Antigen Specificity

city: Nashville

first: Ivelin

last: Georgiev

state: TN

address: 11475 MRB IV 2213 Garland Ave

department: Pathology, Microbiology, and Immunology

institute: Vanderbilt University Medical Center

summary: Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, and largely unique, antigen specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences, but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large dataset mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against twenty viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3/CDRL3 identity threshold that defines whether these B cells may share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

overall_design: Antigen specific B cells  were sorted from human PBMCs using the standard LIBRA-seq pipeline (Shiakolas & Setliff et al., Cell 2019). Cells were distributed into microfluidic droplets containing unique cell-specific oligonucleotides using the 10X Genomics Chromium Controller. cDNA libraires from single cells were further amplified with BCR enrichment primers using the 10X Genomics VDJ protocol.

first: Alexandra

last: Abu-Shmais

first: Matthew

last: Vukovich

first: Perry

last: Wasdin

first: Yukthi

last: Suresh

first: Toma

last: Marinov

first: Scott

last: Rush

first: Rebecca

last: Gillespie

first: Rajeshwer

middle: S

last: Sankhala

first: Misook

last: Choe

first: M

last: Joyce

first: Masaru

last: Kanekiyo

first: Jason

last: McLellan


title: Identification of a DNA methylation signature in whole blood of newborn guinea pigs and human neonates following antenatal betamethasone exposure

status: Public on Aug 22 2024

city: Toronto

first: Stephen

last: Matthews

address: 1 King's College Circle, Rm 3302

institute: University of Toronto

summary: We investigated the effect of antenatal corticosteroid exposure (ACS) on the DNA methylation patterns in the whole blood of female guinea pig offspring. Guinea pig dams were treated with saline or betamethasone on gestational days 50/51. Whole blood was collected from newborn offspring (post-natal day 1) and dried on blood spot cards. Methylation assessment using genomic DNA was performed with reduced representation bisulfite sequencing techniques.

overall_design: Differential DNA methylation in the whole blood (dried blood spots) of newborn female guinea pig offspring was assessed to compare profiles between Betamethasone and saline control exposures. n=7 per treatment condition, total of 14.

first: Kim

last: Bona

first: Kostaki

last: Alice

first: McClymont

last: Sarah

first: Matthews

last: Stephen G.


title: Inter-organ transmission of hepatocellular senescence induces multi-organ dysfunction through the TGFβ signalling pathway.

first: Thomas

last: Bird

summary: Cellular senescence is associated with aging but also impacts various physiological and pathological processes such as embryonic development and wound healing. Factors secreted by senescent cells can affect their microenvironment, including  local spreading of senescence. Acute severe liver disease is associated with hepatocyte senescence and frequently progresses to multi-organ failure. Why the latter occurs is poorly understood however, the presence of hepatic senescence is associated with poor prognosis and extrahepatic organ failure in acute liver disease. Here, using genetic mouse models of hepatocyte-specific senescence, we demonstrate senescence development in extrahepatic organs and associated organ dysfunction in response to liver senescence. In patients with acute indeterminate hepatitis, the extent of hepatocellular senescence predicts the occurrence of extrahepatic dysfunction, need for liver transplantation and mortality. We identify the Transforming Growth Factor β (TGFβ) pathway as a critical mediator of systemic spread of senescence and TGFβ inhibition blocks senescence transmission to other organs preventing renal dysfunction. Our results highlight the systemic consequences of organ-specific senescence which, independent of aging, contributes to multi-organ dysfunction.

overall_design:
An adeno-associated viral (AAV) vector system is used to express the mutant KRasG12D in the hepatocytes of mice. This results in liver senescence, which is followed by senescence in the kidney.
Bulk RNA-Seq was used to determine the transcriptional differences between control KRas wildtypes and mutant KRasG12D. 5 controls and 5 wildtypes from liver and kidney tissue were used.
The TruSeq Stranded mRNA kit was used and the samples sequenced on a NextSeq 500. Raw fastq.gz where quality trimmed with FastP and aligned to the GRCm38.92 genome with HiSat2. The reads where summarised using Feature Counts.

first: Christos

last: Kiourtis

first: Maria

last: Terradas-Terradas

first: Lucy

last: Gee

first: Stephanie

last: May

first: Anastasia

last: Georgakapoulou

first: Amy

last: Collins

first: Miryam

last: Müller

first: Eoin

last: O'Sullivan

first: David

last: Baird

first: Mohsin

last: Hassan

first: Ee

last: Ta

first: Cornelius

last: Engelmann

first: Fausto

last: Andreola

first: Ya-Ching

last: Hsieh

first: Lee

last: Reed

last: Borthwick

first: Colin

last: Nixon

first: William

last: Clark

first: Robin

last: Shaw

first: Peter

last: Hanson

last: Sumpton

first: Gillian

last: Mackay

first: Toshi

last: Susuki

first: Arafath

middle: K

last: Najumudeen

first: Andrew

last: Campbell

first: Simon

last: Barry

first: Alberto

last: Quaglia

first: Christopher

last: Morris

first: Fiona

last: LeBeau

first: Owen

last: Sansom

last: Ferenbach

first: Kristina

last: Kirschner

first: Rajiv

last: Jalan

last: Oakley


title: Single cell transcriptional profiles of endothelial cells  sorted from the hearts with ablation of Wnt/beta-catenin signaling activating cardiomyocytes and without ablation [scRNA-seq]

first: Ayano

last: Chiba

summary: To understand how Wnt/β-catenin signaling-activated cardiomyocytes (β-cat ON CMs) promote coronary vessel development, we performed single-cell RNA sequencing of endothelial cells (ECs) sorted from the hearts ablated β-cat ON CMs and those from control hearts, respectively. Our analyses indicated that β-cat ON CMs regulates coronary vessel development by promoting arterialization, a step in the transition of endocardial ECs to coronary ECs.

overall_design: To induce nitroreductase-metronidazole (NTR-MTZ)-dependent ablation of β-cat ON CMs, we treated 5 mM MTZ or DMSO to Tg(fli1:EGFP);Tg(cryaa:EGFP, myl7:GAL4db-TCFΔC);(5xUAS:NTR-mCherry) fish every other day for 4 weeks from 1 month-old. We isolated live GFP+ cells by FACS sorting from the fish-treated MTZ or DMSO, respectively. Barcoded single-cell cDNA libraries were prepared using the Chromium Next GEM single cell 3’reagent kits v3.1 and sequenced using Illumina NextSeq 500.

first: Moe

last: Fukumoto

first: Manabu

last: Shirai

first: Naoki

last: Mochizuki


title: Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma [CRISPR/Cas9 screen]

city: Badalona

first: David

last: Corujo

address: Ctra de Can Ruti, Camí de les Escoles

institute: Josep Carreras Leukaemia Research Institute

summary: Childhood neuroblastoma with MYCN-amplification is classified as high-risk and often relapses after intensive treatment regimen. Immune checkpoint blockade therapy shows limited efficacy in neuroblastoma patients and the cancer intrinsic immune regulatory network is poorly understood. Here, we leveraged genome-wide CRISPR/Cas9 screens in a human co-culture system and identified H2AFY as a resistance gene to nivolumab. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverted in vivo resistance to PD-1 blockade by eliciting concurrent activation of the adaptive and innate immunity. Analysis of single-cell RNA sequencing datasets revealed that H2AFY mRNA was enriched in adrenergic cancer cells and was associated with patient survival. Mapping of the epigenetic and translational landscape demonstrated that H2afy deletion promoted cell transition to a malignant mesenchymal-like state. With a multi-omics approach, we uncovered H2AFY-associated genes that were functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell state and immunogenicity in high-risk human neuroblastoma.

overall_design: We leverage genome-wide CRISPR/Cas9 screen in IMR32 neuroblastoma cells to identify resistance genes to nivolumab treatment, a clinically approved PD-1 blocking antibody.

first: Divya

last: Nagarajan

first: Ginte

last: Kutkaite

first: Michael

last: Menden

first: Yumeng

last: Mao


title: Transcriptional profiles of Wnt/beta-catenin signaling activated cardiomyocytes and the others in the atrioventricular canal of zebrafish heart [RNA-seq]

summary: To understand the function of Wnt/β-catenin signaling-activated cardiomyocytes (β-cat ON CMs), we performed RNA-Seq on β-cat ON and OFF CMs at the atrioventricular canal (AVC) in the heart, respectively. Our analyses indicated that β-cat ON CMs upregulate genes related to blood vessel development.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for β-cat ON CMs and β-cat OFF CMs.


title: VE-1 requires the WC-1 photoreceptor for light-dependent inhibition of perithecial developmentin Neurospora crassa

city: Sevilla

first: Gabriel

last: Gutierrez

address: PO BOX 1095

department: Genetics

institute: University of Seville

summary: The observation that light led to a large reduction in the number of perithecia in the ∆ve-1 strain suggested that WC-1, the photoreceptor component of the WCC transcription factor complex, may participate in this regulation. Therefore, we analyzed the sexual phenotype of a double mutant with deletions in ve-1 and wc-1

overall_design: Pairwise comparisons of expression profiles obtained from RNA extracted from sexual tissue of the ve-1 mutant, the white-collar mutant and the double mutant vs. the wild-type strain in dark and light

first: Sara

last: Cea-Sánchez

last: Martín-Villanueva

last: Gutiérrez

last: Cánovas

first: Luis

last: Corrochano


title: EP152R-Mediated Endoplasmic Reticulum Stress Contributes to African Swine Fever Virus Infection via the PERK-eIF2α Pathway

city: Beijing

first: Rui

last: Liang

address: Tsinghua  University, Haidian  District

institute: Tsinghua university

summary: African swine fever virus (ASFV) is a large, icosahedral, double-stranded DNA virus in the Asfarviridae family and the causative agent of African swine fever (ASF). ASFV causes a hemorrhagic fever with high mortality rates in domestic and wild pigs. ASFV contains an open reading frame named EP152R, previous research has shown that EP152R is an essential gene for virusrescue in swine macrophages. However, the detailed functions of ASFV EP152R remain elusive. Herein, we demonstrate that EP152R, a membrane protein located in the endoplasmic reticulum (ER), induces ER stress and swelling, triggering the PERK/eIF2α pathway and broadly inhibiting host protein synthesis in vitro. Additionally, EP152R strongly promotes immune evasion, reduces cell proliferation, and alters cellular metabolism. These results suggest that ASFV EP152R plays a critical role in the intracellular environment, facilitating viral replication. Furthermore, virus-level experiments have shown that the knockdown of EP152R or PERK inhibitors efficiently affects viral replication by decreasing viral gene expression. In summary, these findings reveal a series of novel functions of ASFV EP152R and have important implications for understanding host-pathogen interactions.

overall_design: We compared the differential expression of RNAs in 293T cells transinfected with PCAGGS and EP152R using RNA-Seq.

first: Yanan

last: Fu

first: Guosheng

last: Li

first: Guiqing

last: Peng


title: Synergistic Effects of Fructose and Food Preservatives on Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): From Gut Microbiome Alterations to Hepatic Gene Expression

city: Novy Hradek

first: Tomas

last: Hrncir

address: Doly 183

department: Laboratory of Gnotobiology

country: Czech Republic

institute: Institute of Microbiology of the Czech Academy of Sciences

summary:
This study investigates the synergistic effects of fructose and common food preservatives (sodium benzoate, sodium nitrite, and potassium sorbate) on the development of non-alcoholic fatty liver disease (NAFLD) using a human-microbiota-associated mouse model.
We examined changes in hepatic gene expression through microarray analysis to understand the molecular mechanisms underlying NAFLD progression in response to these dietary factors.

overall_design: Mice were divided into five groups: control, fructose-only, fructose+benzoate, fructose+nitrite, and fructose+sorbate. After 11 weeks of treatment, liver samples were collected for gene expression analysis using GeneChip Clariom S arrays. Each group consisted of 3 biological replicates.

first: Eva

last: Trckova

first: Lucia

last: Hrncirova


title: Manipulating mitochondrial reactive oxygen species alters survival in unexpected ways in a Drosophila Cdk5 model of neurodegeneration

city: Bethesda

first: Kory

middle: R

last: Johnson

address: 10/3B01, 9000 Rockville Pike

department: DIR IT & Bioinformatics

institute: NINDS/NIH

summary: Reactive oxygen species (ROS) are associated with aging and neurodegeneration, but the significance of this association remains obscure. Here, using a Drosophila Cdk5 model of age-related neurodegeneration, we probe this relationship in the pathologically relevant tissue, the brain, by quantifying three specific mitochondrial ROS and manipulating these redox species pharmacologically. Our goal is to ask whether pathology-associated changes in redox state are detrimental for survival, whether they may be beneficial responses to pathology, or whether they are simply covariates of pathology that do not alter viability. We find, surprisingly, that increasing mitochondrial H2O2 correlates with improved survival. We also find evidence that drugs that alter the mitochondrial glutathione redox potential modulate survival primarily through the compensatory effects they induce rather than through their direct effects on the final mitochondrial glutathione redox potential per se. We also find that the response to treatment with a redox-altering drug varies dramatically depending on the age at which the drug is administered, the duration of the treatment, and the genotype of the individual receiving the drug. These data have important implications for the design and interpretation of studies investigating the effect of redox state on health and disease as well as on efforts to modify the redox state to achieve therapeutic goals.

overall_design: 40 samples representing 13-21 heads from Wild-type male Drosophila melanogaster (10 days-old) that have been treated with either vehicle (DMSO), vehicle and 2-AAPA, or vehicle and MitoQ acutely (24 hrs prior to RNA extraction and isolation ) or chronically (10 days).

first: Andrew

last: Wodrich

first: Brent

last: Harris

first: Edward

last: Giniger


title: PML is a novel constitutive component of chromatin domains of enriched repetitive elements and duplicated gene clusters in cancer cells

city: Milan

first: Rosa

last: Bernardi

address: via Olgettina 60

country: Italy

institute: San Raffaele Scientific Institute

summary:
Heterochromatin is a pivotal element in the functional organization of genomes. In our study, we delve into
the heterochromatin pattern of association by the PML (promyelocytic leukemia) protein. By using PML
chromatin immunoprecipitation and sequencing data and comparing computational methodologies to depict
PML chromatin association, we describe PML-associated domains or PADs as large heterochromatic
regions that exhibit similar genomic features across cancer cell lines. We show that PADs are specifically
enriched in non-coding genes, duplicated gene clusters, and repetitive DNA elements. Moreover, we find
enriched binding motifs of KZFPs, which are involved in orchestrating epigenetic repression at repetitive
DNA elements. Hence, our findings suggest that PML conservatively associates to heterochromatic
domains enriched in repetitive DNA elements and duplicated gene clusters in cancer. These findings
contribute to a broader understanding of the complex regulatory framework of genome organization by
heterochromatin in cancer.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for PML in RCC4 cells.

first: Fracassi

last: Cristina

first: Simoni

last: Matilde

first: Marco

last: Morelli

first: Bernardi

last: Rosa

first: Martina

last: Uggè


title: Midkine as a driver of age-related changes and increase in mammary tumorigenesis [scRNA-seq]

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Aging is one of the pivotal risk factors for cancer, notably in breast cancer with diagnosis striking at the average age of 62. However, the intricate mechanisms underlying aging and breast cancer susceptibility remain unclear. In this study, we depicted a comprehensive single-cell landscape of gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) of mammary glands from different aged rats. Mechanically, we revealed midkine, a growth factor secreted by basal epithelial cells, which might mediates the age-related mammary changes, as a validation, we treated young rats with midkine for a month and performed the single-cell transcriptome analysis on those mammary glands. We found midkine could largely mediates the transcriptional shift and hyperproliferation of aged epithelial cell by activating PI3K/AKT-SREBF1 signaling. Furthermore, we find the aging-related accumulation of midkine could largely mediated aging-related changes of mammary gland and promoting the tumorigenesis of breast tumors proved using a well-established Nitroso-N-methylurea (NMU)-induced breast cancer rat model. Our finding identify a promising biomarker and intervention target for both mammary aging and tumorigenesis.

overall_design: Mammary glands were freshly collected from different aged rats (3-, 6-, 12- and 22-month-old) and rats (2-month-old) with one-month midkine treatment. The tissue was digested into single cells, then submitted for single-cell RNA sequencing.

first: Pengze

last: Yan


title: Midkine as a driver of age-related changes and increase in mammary tumorigenesis [scATAC-seq]

overall_design: Mammary glands were freshly collected from different aged rats (3-, 6-, 12- and 22-month-old). The tissue was digested into single cells, then submitted for single-cell ATAC sequencing.


title: Midkine as a driver of age-related changes and increase in mammary tumorigenesis [bulk RNA-seq]

overall_design: For NMU-induced mammary tumor experiment, Fischer rats were treated with midkine protein at 300 ug/kg 3 times/ week for two weeks and followed by a single intraperitoneal injection (i.p.) of 50 mg/kg N-nitroso N-methylurea (NMU). Then, rats were treated with midkine protein for another two weeks with same injection frequency, followed by another single NMU injection. Rats were checked three times/ week, and tumor growth was monitored using caliper measurements. Maximum tumor size burden allowed for rats is 4 cm and this was not exceeded in any of the experiments. All the remaining rats were sacrificed upon the last tumor in midkine treatment group reached the size limit. Rats were euthanized by CO2 inhalation. All the tumors from vehicle and midkine treatment groups were saved for bulk RNA-seq analysis.


title: Midkine as a driver of age-related changes and increase in mammary tumorigenesis

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

overall_design: Refer to individual Series


title: CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing.

city: Strasbourg

first: Todd

middle: L

last: Blevins

address: 12 Rue du Général-Zimmer

country: France

institute: CNRS

summary: Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

overall_design:
Small RNA sequencing of Arabidopsis flower samples from a panel of nrpd1 and clsy mutants, along with three variants of NRPD1 complementation line (WT, Mut9, Mut10). Two biological replicates were included per genotype.

Grant info: T. Blevins  ANR-17-CE20-0004 (Agence nationale de la recherche)
Grant info: T. Blevins  ANR-10-IDEX-0002 (Agence nationale de la recherche)
Grant info: J. A. Law  GM112966         (NIH)

first: Luisa

last: Felgines

first: Bart

last: Rymen

first: Laura

last: Martins

first: Guanghui

last: Xu

first: Calvin

last: Matteoli

first: Christophe

last: Himber

first: Julie

last: Law